CN118892453A - INKT cell modulator liposome compositions and methods of use - Google Patents

Abstract

Provided herein are compositions comprising liposomes comprising compound a

Description

INKT cell modulator liposome compositions and methods of use

Background Art

Natural killer T (NKT) cells are involved in a range of important immune surveillance mechanisms such as host defense against external pathogens, immune tolerance, and malignancy. NKT cells can be further divided into two subsets, type I and type II. Type I NKT cells have received the most attention. These cells are also known as invariant NKT (iNKT) cells because they express a constant α-chain T-cell receptor (TCR; Vα14–Jα18 chain in mice and Vα24–Jα18 chain in humans) that is paired with a more variable β chain. In contrast, type II NKT cells have a diverse TCR repertoire and are less well defined, although one subset has been shown to respond to sulfolipids. The iNKT cell TCR recognizes lipid antigens presented in the context of the non-polymorphic MHC class I protein CD1d. The CD1d molecule has been shown to bind to a range of dialkyl lipids and glycolipids, and subsequent recognition of the CD1d lipid complex by the iNKT cell TCR leads to rapid proliferation and release of a plethora of (pro-inflammatory and regulatory) cytokines. The activation of iNKT cells is an important step in "boosting" the adaptive immune response, which can be achieved through the activation and maturation of dendritic cells (DCs) and B cells through CD40-CD40L interactions, as well as the activation of natural killer (NK) cells after the release of interferon gamma (IFNγ). Since the structure of CD1d ligands has been shown to control the spectrum of cytokines released, it can be found that the development of lipid molecules that promote the specific activation of iNKT cells is very useful in the application of treating various diseases.

Among a range of lipids that bind to CD1d, the glycolipid α-galactosylceramide (α-GalCer) is one of the most potent. α-GalCer is a derivative of agelasphins, naturally occurring glycolipids isolated from the marine sponge Agelas mauritianus. Recognition of the α-GalCer-CD1d complex by the iNKT cell TCR leads to the secretion of a range of cytokines and the initiation of a strong immune response.

Although α-GalCer remains one of the most potent iNKT cell agonists and shows promise in treating a variety of pathologies, it has proven difficult to use this molecule broadly as an effective therapeutic agent, at least as a direct activator of iNKT cells: α-GalCer-mediated activation of iNKT cells not only results in the secretion of T helper 1 (Th1) (e.g., IFN-γ) and T helper 2 (Th2) (e.g., interleukin-4 (IL-4)) cytokines, thereby generating a mixed immune response, but more importantly, overstimulates iNKT cells, which may cause them to enter a state of long-term anergy, i.e., no response to subsequent α-GalCer stimulation and preferential production of IL-4, which is detrimental to long-term treatment. If multiple dose regimens are required, the loss of circulating levels of iNKT cells may represent a significant efficacy limitation for iNKT cell-based therapies. Therefore, there is a need for iNKT cell activator compositions with effective delivery for clinical applications.

Novel iNKT cell agonists are described in International Patent Application No. PCT/EP2012/074140 filed on November 30, 2012, and International Publication No. WO 2013/079687, entitled "INKT Cell Modulators and Methods of Using the Same". One of these compounds, threitolceramide-(IMM60), has been shown to be very effective in amplifying antigen-specific T cell responses. See Jukes et al., Non-glycosidic compounds can stimulate both human and mouse iNKT cells. Eur J Immunol, 2016; 46(5): 1224-1234. Having a threitol moiety in the polar head of IMM60 would result in a more stable iNKT agonist, thereby increasing its bioavailability, which in turn would increase maturation and stimulation of antigen-presenting DCs.

There is a need for improved formulations of IMM60 and related compounds, as well as improved therapeutic combinations and regimens using these iNKT cell antagonists.

Summary of the invention

The present disclosure has many aspects, including liposomes; compositions (such as pharmaceutical compositions) containing liposomes; methods of preparing liposomes and compositions; methods of evaluating and optimizing liposome compositions; and methods of using the compositions, including therapeutic and preventive methods for preventing, ameliorating, treating and curing diseases; and methods of modulating the immune response of mammalian subjects. Exemplary embodiments of the invention are summarized or defined by the following numbered paragraphs:

1. A liposome, comprising compound A:

(A)

wherein n is 1 (IMM60), 2 (IMM70) or 3 (IMM80), or a salt, ester, solvate or hydrate thereof, and two or more lipids or salts thereof;

Wherein the two or more lipids comprise:

(a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG);

(b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol);

(c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB);

(d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC);

(e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); or,

(f) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL).

2. The liposome of Example 1, wherein n is 1 (IMM60).

3. The liposome of embodiment 1 or 2, wherein Compound A is present in an amount of about 0.1 wt % to about 20 wt % based on the total weight of the liposome.

4. The liposome of embodiment 1 or 2, wherein Compound A is present in an amount of about 0.5 wt % to about 15 wt %, or about 1 wt % to about 12 wt %, or about 5 wt % to about 12 wt %, based on the total weight of the liposome.

5. The liposome of any one of embodiments 1 to 4, wherein the two or more lipids comprise from about 75 wt % to about 99.9 wt %, or from about 80 wt % to about 98 wt %, or from about 80 wt % to about 95 wt %, or from about 85 wt % to about 95 wt %, based on the gross weight of the liposome.

6. The liposome of any one of embodiments 1 to 5, wherein the mass ratio of Compound A to the two or more lipids is from about 1:5 to about 1:20, or from about 1:7 to about 1:15, or from about 1:7 to about 1:12, or about 1:9.

7. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).

8. The liposome of embodiment 7, wherein the DSPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 60 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome.

9. The liposome of embodiment 7 or 8, wherein the DSPG is present in an amount of about 20 wt % to 60 wt %, or about 30 wt % to about 50 wt %, or about 35 wt % to about 45 wt %, based on the total weight of the liposome.

10. The liposome of any one of embodiments 7-9, wherein the liposome further comprises cholest-5-ene-3β-ol (CHOL).

11. The liposome of embodiment 10, wherein the CHOL is present in an amount of about 1 wt % to about 20 wt %, or about 5 wt % to about 15 wt %, or about 8 wt % to about 12 wt %, based on the total weight of the liposome.

12. The liposome of embodiment 10 or 11, wherein the DSPC is present in an amount of about 40 wt % to about 50 wt %, the DSPG is present in an amount of about 30 wt % to about 40 wt %, and the CHOL is present in an amount of about 5 wt % to about 15 wt %.

13. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol).

14. The liposome of embodiment 13, wherein the POPC is present in an amount of about 40 wt % to 80 wt %, or about 45 wt % to about 65 wt %, or about 50 wt % to about 60 wt %, based on the total weight of the liposome.

15. The liposome of embodiment 13 or 14, wherein the DC-Chol is present in an amount of about 15 wt % to 55 wt %, or about 20 wt % to about 50 wt %, or about 25 wt % to about 35 wt %, based on the total weight of the liposome.

16. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB).

17. The liposome of embodiment 16, wherein the POPC is present in an amount of about 40 wt % to 80 wt %, or about 45 wt % to about 65 wt %, or about 50 wt % to about 60 wt %, based on the total weight of the liposome.

18. The liposome of embodiment 1 or 17, wherein the DC-Chol is present in an amount of about 15 wt % to 55 wt %, or about 20 wt % to about 50 wt %, or about 30 wt % to about 40 wt %, based on the total weight of the liposome.

19. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC).

20. The liposome of embodiment 19, wherein the EPG is present in an amount of about 40 wt % to 80 wt %, or about 55 wt % to about 75 wt %, or about 60 wt % to about 70 wt %, based on the total weight of the liposome.

21. The liposome of embodiment 19 or 20, wherein the EPC is present in an amount of about 10 wt % to 50 wt %, or about 15 wt % to about 40 wt %, or about 15 wt % to about 30 wt %, based on the total weight of the liposome.

22. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

23. The liposome of embodiment 22, wherein the POPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 65 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome.

24. The liposome of embodiment 22 or 23, wherein the DOTAP is present in an amount of about 20 wt % to 50 wt %, or about 25 wt % to about 45 wt %, or about 35 wt % to about 45 wt %, based on the total weight of the liposome.

25. The liposome of any one of embodiments 1 to 6, wherein the two or more lipids comprise 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL).

26. The liposome of embodiment 25, wherein the DMPG is present in an amount of about 40 wt % to 80 wt %, or about 50 wt % to about 75 wt %, or about 60 wt % to about 70 wt %, based on the total weight of the liposome.

27. The liposome of embodiment 25 or 26, wherein the CHOL is present in an amount of about 10 wt % to 50 wt %, or about 15 wt % to about 35 wt %, or about 15 wt % to about 30 wt %, based on the total weight of the liposome.

28. The liposome of any one of embodiments 1 to 27, wherein the liposome further comprises a liposome buffer.

29. The liposome of embodiment 28, wherein the pH of the liposome buffer is about 7, or about 6.3 to about 6.7.

30. The liposome of embodiment 29, wherein the liposome buffer comprises sodium chloride and sodium phosphate.

31. The liposome of any one of embodiments 28-30, wherein the liposome buffer further comprises sucrose.

32. The liposome of any one of embodiments 1 to 31, wherein the liposome further comprises at least one antigen.

33. The liposome of embodiment 32, wherein the at least one antigen comprises a member selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, tumor antigens, and mixtures thereof.

34. The liposome of embodiment 32 or 33, wherein the at least one antigen comprises at least one tumor antigen.

35. The liposome of embodiment 34, wherein the tumor antigen is selected from the group consisting of:

(a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB 33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE- C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosine enzyme, melan-A, XAGE,

(b) an antigenic fragment of any one of (a), and

(c) A mixture of any one of (a) and/or (b).

36. The liposome of any one of embodiments 31-35, wherein the antigen is present in the liposome in an amount of about 1 g antigen to 1-20 g compound A.

37. The liposome of any one of embodiments 1 to 36, further comprising at least one therapeutic agent.

38. A composition comprising the liposome of any one of embodiments 1-37 and a pharmaceutically acceptable diluent, excipient, adjuvant or carrier.

39. The composition of embodiment 38, wherein the average diameter of the liposome is less than 200 nm.

40. The composition of embodiment 38, wherein the average diameter of the liposomes is in the range of about 50 nm to 200 nm, or about 50 nm to about 170 nm, or about 75 nm to about 145 nm, or about 90 nm to about 130 nm.

41. The composition of embodiment 38, wherein the average diameter of the liposome is 110 nm ± 60 nm, 110 nm ± 40 nm or 110 nm ± 20 nm.

42. The composition of any one of embodiments 38 to 41, wherein the liposome has a polydispersity index (PdI) of less than or equal to about 0.40, or less than or equal to about 0.35, or less than or equal to about 0.30, or less than or equal to about 0.25, or less than or equal to about 0.20, or less than or equal to about 0.15.

43. The composition of any one of embodiments 38 to 42, wherein the composition further comprises at least one antigen.

44. The composition of embodiment 43, wherein at least one of the antigens comprises a member selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, tumor antigens, and mixtures thereof.

45. The composition of embodiment 43 or 44, wherein at least one of the antigens comprises at least one tumor antigen.

46. The composition of embodiment 45, wherein the tumor antigen is selected from the group consisting of:

(a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB 33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE- C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosine enzyme, melan-A, XAGE,

(b) an antigenic fragment of any one of (a), and

(c) A mixture of any one of (a) and/or (b).

47. The composition of embodiment 45, wherein the tumor antigen comprises NY-ESO-1 or an antigenic fragment thereof.

48. The composition of any one of embodiments 38 to 47, further comprising at least one therapeutic agent.

49. The composition of embodiment 48, wherein at least one therapeutic agent is selected from the group consisting of an immunomodulator, a Toll-like receptor agonist, a Nod ligand, an antiviral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immunotherapeutic agent, a chemotherapeutic agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and a combination thereof.

50. A method of stimulating an immune response in a mammalian subject, the method comprising administering to the subject the composition of any one of embodiments 38 to 49.

51. The method of embodiment 50, further comprising, prior to the administering step, the step of heating the composition for a time and at a temperature sufficient to reduce the average particle size or PdI of the liposome composition.

52. The method of embodiment 50, further comprising, prior to the applying step, the step of heating the composition at a temperature of about 55°C to about 65°C for 5-15 minutes.

53. The composition of any one of embodiments 38 to 49, for use in stimulating an immune response in a mammalian subject.

54. Use of the composition of any one of embodiments 38 to 49 for stimulating an immune response in a mammalian subject.

55. The liposome of any one of embodiments 1 to 37 or the composition of any one of embodiments 38 to 49 for use in the preparation of a medicament for stimulating an immune response in a mammalian subject.

56. The composition or use of any one of embodiments 53 to 55, wherein the composition is subjected to a heating step before being used to stimulate an immune response.

57. The method or use of any one of embodiments 50-56, wherein the mammalian subject is a human.

58. The method or use of any one of embodiments 50 to 57, wherein the mammalian subject has cancer.

59. A method of treating a mammalian subject having cancer, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of embodiments 38 to 49.

60. The method or use of embodiment 58 or 59, wherein the composition is administered by subcutaneous, intravenous or intratumoral injection.

61. The method of embodiment 50 or 60, further comprising, prior to the administering step, the step of heating the composition for a time and at a temperature sufficient to reduce the average particle size or PdI of the liposome composition.

62. The liposome of any one of embodiments 1 to 37 or the composition of any one of embodiments 38 to 49 for use in treating cancer in a mammalian subject.

63. The liposome of any one of embodiments 1 to 37 or the composition of any one of embodiments 38 to 49 for use in the preparation of a medicament for treating cancer in a mammalian subject.

64. The composition or use of any one of embodiments 61 to 62, wherein the composition is subjected to a heating step before being used to stimulate an immune response.

65. The method or use of any one of embodiments 58 to 64, wherein the cancer is selected from the group consisting of basal cell carcinoma, breast cancer, leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell carcinoma, melanoma, and combinations thereof.

66. The method or use of any one of embodiments 58 to 64, wherein the cancer comprises melanoma.

67. The method or use of any one of embodiments 58 to 64, wherein the cancer comprises non-small cell lung cancer.

68. The method or use of any one of embodiments 58 to 67, further comprising administering to the subject or using at least one further treatment or therapy selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, and radiation therapy.

69. The method or use of any one of embodiments 58-68, further comprising administering or using at least one checkpoint inhibitor to the subject.

70. The method or use of Example 69, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor.

71. The method or use of embodiment 70, wherein the immune checkpoint inhibitor comprises at least one PD-1 inhibitor selected from Pembrolizumab, Nivolumab, Cemiplimab and Spartalizumab.

72. The method or use of embodiment 70, wherein the immune checkpoint inhibitor comprises at least one PD-L1 inhibitor selected from atezolizumab, avelumab and durvalumab.

73. The method or use of embodiment 70, wherein the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab.

74. The method or use of any one of embodiments 68-73, wherein the composition and the further treatment or therapy are administered simultaneously.

75. The method or use of any one of embodiments 68-73, wherein the composition and the further treatment or therapy are administered separately.

76. A method for preparing liposomes, the liposomes comprising compound A:

wherein n is 1 (IMM60), 2 (IMM70) or 3 (IMM80), or a salt, ester, solvate or hydrate thereof, and two or more lipids or salts thereof; the method comprising:

(A) mixing compound A and two or more lipids to form multilamellar vesicles,

Wherein the two or more lipids comprise:

(a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG);

(b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol);

(c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyl dioctadecyl

Ammonium bromide (DDAB);

(d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC);

(e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); or

(f) 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL), and

(B) Multilamellar vesicles are extruded through the membrane to form liposomes.

77. The method of embodiment 76, further comprising (c) purifying the liposomes.

78. A method as described in Example 77, wherein the purification comprises filtration.

79. A method as described in Example 77, wherein the purification comprises ultrafiltration, centrifugation, dialysis, diafiltration or tangential flow filtration.

80. The method of embodiment 78, comprising filtering with a filter having a pore size of 400 nm, or less than about 350 nm, or less than about 300 nm, or less than about 250 nm, or less than about 220 nm, or less than about 200 nm, or less than about 150 nm.

81. The method of embodiment 78, comprising filtering with a filter having a pore size of about 50 nm to about 250 nm, or about 50 nm to about 150 nm, or about 60 nm to about 100 nm, or about 80 nm.

82. The method of embodiment 78, comprising filtering with a filter having a molecular weight cutoff (MWCO) of 1000 kD, or 800 kD, or 600 kD, or 500 kD, or 400 kD, or 300 kD, or 250 kD.

83. The method of any one of embodiments 7-82, wherein n is 1 (IMM60).

84. The method of any one of embodiments 7-83, wherein Compound A is present in an amount of about 0.1 wt % to about 20 wt %, or about 0.5 wt % to about 15 wt %, or about 1 wt % to about 12 wt %, or about 5 wt % to about 12 wt %, based on the total weight of the liposome.

85. The method of any one of embodiments 7-84, wherein the two or more lipids comprise from about 75 wt % to about 99.9 wt %, or from about 80 wt % to about 98 wt %, or from about 80 wt % to about 95 wt %, or from about 85 wt % to about 95 wt %, based on the gross weight of the liposome.

86. The method of any one of embodiments 7-81, wherein the mass ratio of Compound A to two or more lipids is in the range of about 1:5 to about 1:20, or about 1:7 to about 1:15, or about 1:7 to about 1:12, or about 1:9.

87. The method of any one of embodiments 7-86, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).

88. The method of embodiment 87, wherein the DSPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 60 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome.

89. The method of embodiment 87 or 88, wherein the DSPG is present in an amount of about 20 wt % to 60 wt %, or about 30 wt % to about 50 wt %, or about 35 wt % to about 45 wt %, based on the total weight of the liposome.

90. The method of any one of embodiments 7-84, wherein the two or more lipids further comprise CHOL.

91. A method as described in any of Examples 7-90, wherein step (A) further comprises mixing at least one antigen with the compound A and the lipid.

92. A method as described in Example 91, wherein the at least one antigen comprises a viral antigen, a bacterial antigen, a fungal antigen and/or a tumor antigen.

93. The method of embodiment 91 or 92, wherein the at least one antigen comprises at least one tumor antigen.

94. The liposome of embodiment 93, wherein the tumor antigen is selected from the group consisting of:

(a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB 33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE- C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosine enzyme, melan-A, XAGE,

(b) an antigenic fragment of any one of (a), and

(c) A mixture of any one of (a) and/or (b).

95. A method as described in any one of embodiments 7-94, wherein step (A) further comprises mixing at least one therapeutic agent with the lipid and the compound A.

96. A liposome prepared by the method of any one of embodiments 76-95.

97. A method for treating a liposome preparation, the method comprising

(a) providing a liposome composition as described in any one of Examples 38-49; and

(b) heating the composition for a period of time and at a temperature sufficient to reduce the average particle size or PdI of the liposome composition.

98. A method for treating a liposome preparation, the method comprising

(a) providing a liposome composition as described in any one of Examples 38-49; and

(b) heating the composition at a temperature of about 55°C to about 65°C for 5-15 minutes.

99. The method of embodiment 97 or 98, wherein the liposome composition provided in (a) has been frozen, stored at below 0°C or below -20°C, or freeze-dried and reconstituted.

The foregoing summary is not intended to limit every aspect of the present disclosure, and additional aspects are described in other sections such as the detailed description. The entire document is intended to be a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, paragraph or section of this document.

In addition to the foregoing, the present disclosure also includes (as additional aspects) all embodiments of the present disclosure that are in any way narrower in scope than the variations specifically described above. With respect to aspects of the present disclosure described or claimed as "one/kind", unless the context clearly requires a more restricted meaning, these terms mean "one or more". With respect to one or more elements described as within a set, all combinations within the set are considered to be combined inventions. If aspects of the present disclosure are described as "comprising" features, it is also envisioned that embodiments "consist of" or "consist of mainly" the features.

Aspects of the disclosure described as methods of treatment should also be understood to include the first or subsequent "medical use" aspect of the disclosure or "Swiss use" of a composition for the preparation of a medicament for treating the same disease or condition.

For the combination described herein, multiple embodiments are envisioned. For example, some aspects of the present disclosure are described as a method of treatment (or medical use) of combining two or more compounds or agents, whether it is administered alone (sequentially or simultaneously) or in combination (co-formulated or mixed). For each aspect described in this manner, the present disclosure also includes a composition comprising two or more compounds or agents co-formulated or mixed with each other; and the present disclosure also includes a kit (test kit) or a unit dose, which contains two or more compounds/agents packaged together but not mixed. Optionally, such a composition, kit or dosage also includes one or more carriers, which are mixed with one or two agents, or co-packaged for preparation before being applied to a subject. Vice versa: some aspects of the present disclosure are described herein as compositions that can be used for treatment and contain two or more therapeutic agents. Equivalent methods and uses are specifically contemplated.

The present disclosure also relates to materials and methods for improving the effectiveness of cancer treatment using checkpoint inhibitors, including materials and methods for expanding the range of patients or cancers that can be treated with checkpoint inhibitors and/or improving the effectiveness of treatment.

Although one or more applicants have invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass prior art within their scope. Therefore, in the event that statutory or judicially recognized prior art within the scope of the claims is brought to the attention of the applicants by a patent office or other entity or individual, one or more applicants reserve the right to exercise amendment rights under applicable patent law to redefine the subject matter of such claims to specifically exclude such prior art or obvious variations of the statutory prior art from the scope of such claims. Variations of the present disclosure defined by such amended claims are also intended to be aspects of the present invention. Additional features and variations of the present invention will be apparent to those skilled in the art in light of the entirety of this application, and all such features are intended to be aspects of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected into wild-type C57 BL/ or C57 BL/CD1d KO-/- (NKT cell deficient) mice, wherein IFNγ levels were measured in serum 18 hours after injection.

FIG. 1B shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected into wild-type C57 BL/ or C57 BL/CD1d KO-/- (NKT cell deficient) mice, where CD8 levels on dendritic cells (CD11c ⁺ MHC class II ⁺ ) were measured.

Figure 1C shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected in a soluble form (5 ng) into wild-type C57 BL/ or C57 BL/CD1d KO-/- (NKT cell deficient) mice, where PD-L1 levels on dendritic cells (CD11c ⁺ MHC class II ⁺ ) were measured.

FIG. 1A-1C Abbreviations: DC = dendritic cell; Geo MCF = geometric mean channel fluorescence; IFN-γ = interferon-γ; KO = knockout; MHC = major histocompatibility complex; PD-L1 = programmed cell death receptor ligand 1.

Figure 1D shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected into wild-type C57 BL/ (ten bars on the left) or C57 BL/CD1d KO-/- (NKT cell deficient) mice (four bars on the right), where CD40 levels on dendritic cells (CD11c ⁺ MHC II ⁺ class) were measured. The CD40 levels of mice administered with a composition disclosed herein were compared with the CD40 levels of mice administered with dissolved IMM60 (sol IMM60) not formulated in liposomes.

Fig. 1E shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected into wild-type C57 BL/(ten bars on the left) or C57 BL/CD1d KO-/-(NKT cell deficiency) mice (four bars on the right), wherein CD8 levels on B cells were measured. The CD8 levels of mice administered with a composition disclosed herein were compared with the CD8 levels of mice administered with dissolved IMM60 (sol IMM60) not formulated in liposomes.

Figure 1F shows the results of an experiment in which 0.1 ng, 1 ng and 5 ng of IMM60 formulated in a liposome composition disclosed herein were intravenously injected into wild-type C57 BL/ (ten bars on the left) or C57 BL/CD1d KO-/- (NKT cell deficient) mice (four bars on the right), where PDL-1 levels on B cells were measured.

Figures 2-4 summarize the results of experiments in which 0.01ng, 0.1ng and 1ng of IMM60 formulated in the liposome composition disclosed herein were intravenously injected into wild-type C57 BL/ or C57 BL/CD1d KO-/- (NKT cell deficiency) mice, wherein the levels of IFNγ, IL-12p70, IL-2, IL-1β, IL6, KC/GRO, TNF-α, IL-10, IL-4 and IL-5 were measured 4 hours, 18 hours and 72 hours after injection. The levels of these cytokines and markers after injection of the liposome composition disclosed herein were compared with the levels after injection of dissolved IMM60 (IMM60 control, not formulated in liposomes) or vehicle control. Figure 2 summarizes the experimental data performed with IMM60 formulated in POPC/DDAB liposomes. Figure 3 summarizes the experimental data performed with IMM60 formulated in DSPC/DSPG liposomes. Figure 4 summarizes data from experiments performed with IMM60 formulated in POPC/DC-Chol liposomes.

Figure 5 shows the results of experiments in which 10 ng, 100 ng, 1000 nm and 5000 ng IMM60 formulated in the liposome compositions disclosed herein were injected intravenously into wild-type C57 BL/6 (left to right for each formulation), where IFNγ levels were measured in serum 18 hours after injection.

Figure 6 shows the results of an experiment in which mice were treated with soluble IMM60 or liposomal IMM60 formulations disclosed herein. IFN-γ was measured 18 hours after treatment.

7 shows the measurement of CD8 dendritic cells in mice treated with the liposomal IMM60 formulations disclosed herein compared to mice treated with soluble (free) IMM60 or vehicle or untreated controls.

8 shows the measurement of antigen-specific T cells in mice after seven days of treatment with an IMM60 liposome (lip) formulation disclosed herein and an ovalbumin peptide antigen, compared to treatment with soluble IMM60 and the antigen.

Figure 9A shows the results of a preventive dose-response experiment to evaluate

The ability of DPSC/DSPG/IMM60 liposomes to inhibit lung metastatic nodules. Three days before the injection of cells from a melanoma tumor cell line, mice were injected with a liposomal IMM60 formulation at a specified concentration. Figure 9B shows the results of a similar therapeutic dose-response experiment. Three days after the injection of cells from a melanoma tumor cell line, mice were injected with a liposomal IMM60 formulation at a specified concentration. Abbreviations: NS = not significant; * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Figure 10 shows the results of a dose response experiment in mice treated with the IMM60 liposome formulation disclosed herein alone or in combination with an anti-PD-1 antibody in a separate subcutaneous melanoma mouse model. IFN-γ was measured 18 hours after the first administration of the treatment dose. Abbreviations: Ab = antibody; IFNγ = interferon gamma; PD-1 = programmed cell death protein-1.

FIG. 11 shows the results of a tumor growth experiment in mice treated with the liposomal formulation disclosed herein, an anti-PD-1 blocking antibody, or both in a subcutaneous melanoma mouse model.

Figures 12A and 12B show the results of longitudinal tumor growth kinetics in a subcutaneous CT-2 tumor model in mice treated with liposomal formulations disclosed herein, anti-PD-1 blocking antibodies, or both. Figure 12C shows the results of longitudinal tumor growth kinetics at day 14 in a subcutaneous CT-2 tumor model in mice treated with liposomal formulations disclosed herein, anti-PD-1 blocking antibodies, or both. Abbreviations: IFN = interferon; PD-1 = programmed cell death protein-1. Figure 12D shows the measurement of interferon gamma in blood drawn from mice at t = 18 hours in the longitudinal study.

Figure 13 depicts the results of a study (Example 9) in which PDL-1 expression was measured following exposure of melanoma B16f10 cells to varying concentrations of single agents and combinations of agents.

Figure 14 depicts the effects of different concentrations of IMM60, alone or in combination with anti-PD-1 antibodies, on T cells in B16f10:spleen cell co-cultures. (Example 9.)

15A and 15B depict an overview of the Phase 1 and Phase 2 clinical protocols in Example 13.

DETAILED DESCRIPTION

The liposome disclosed herein comprises the following compound A,

Wherein, n is 1 (IMM60), 2 (IMM70) or 3 (IMM80), or its salt, ester, solvate or hydrate, and two or more lipids or salts thereof. Also disclosed is a composition comprising a liposome and a pharmaceutically acceptable diluent, excipient, adjuvant or carrier.

The liposomes provided herein provide a more specific and more effective delivery form for Compound A; and provide stability for the compound. In addition, the liposome formulation is intended to improve the absorption, distribution, metabolism and/or excretion (ADME) of Compound A, particularly for the treatment of cancer tumors. Without being limited to any particular theory, some advantages of the liposome formulations described herein include the EPR effect (enhanced permeability and retention), a longer in vivo half-life, a lower distribution volume, and better tolerability (reducing adverse events).

Compound A (iNKT modulator) or its salt, ester, solvate or hydrate

The compound classified as Compound A herein and the method for preparing the same are described in International Patent Application No. PCT/EP2012/074140 filed on November 30, 2012, the International Publication No. of which is WO

2013/079687, entitled "INKT Cell Modulators and Methods of Using the Same," is incorporated herein by reference in its entirety. See also U.S. Patent No. 9,365,496, which is incorporated herein by reference.

Salts (eg, pharmaceutically acceptable salts) of the disclosed therapeutic agents can be prepared by reacting an appropriate base or acid with a stoichiometric equivalent of the therapeutic agent.

Acids commonly used to form pharmaceutically acceptable salts include inorganic acids (such as hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid) and organic acids (such as p-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, benzenesulfonic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid), as well as related inorganic and organic acids. Thus, such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4- Pharmaceutically acceptable acid addition salts include benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, O-hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and salts formed with organic acids such as maleic acid.

Pharmaceutically acceptable base addition salts can be formed with metals or amines such as alkali metals and alkaline earth metals or organic amines. Pharmaceutically acceptable salts of Compound A can also be prepared with pharmaceutically acceptable cations. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkali metal cations, alkaline earth metal cations, ammonium and quaternary ammonium cations. Carbonates or bicarbonates are also possible. Examples of metals used as cations are sodium, potassium, magnesium, ammonium, calcium or iron, etc. Examples of suitable amines include isopropylamine, trimethylamine, histidine, N,N'-dibenzhydrylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine and procaine.

Similarly, pharmaceutically acceptable derivatives (e.g., esters), metabolites, hydrates, solvates, and prodrugs of therapeutic agents can be prepared by methods well known to those skilled in the art. Therefore, another embodiment provides a composition comprising a prodrug of active compound A. In general, a prodrug is a compound that is metabolized in vivo (e.g., by metabolic transformations such as deamination, dealkylation, deesterification, etc.) to provide an active compound. "Pharmaceutically acceptable prodrug" refers to a compound that is suitable for medicinal use in patients without inappropriate toxicity, irritation, allergic reactions, etc. and is effective for the intended use within the scope of reasonable medical judgment, including pharmaceutically acceptable esters of therapeutic agents and, where possible, zwitterionic forms of therapeutic agents. When used herein, the term "pharmaceutically acceptable ester" refers to an ester that is hydrolyzed in vivo, and includes esters that are easily decomposed in the human body to leave the parent compound or its salt. Suitable ester groups include, for example, ester groups derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic acids, olefinic acids, cycloalkanoic acids, and alkanedioic acids, wherein each alkyl or alkenyl moiety has no more than a plurality of carbon atoms under favorable circumstances. Representative examples of specific esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates. Examples of pharmaceutically acceptable prodrug types are found in Pro-drugs as Novel Delivery Systems by Higuchi and Stella, A.C.S. Monograph Series Vol. 14, and Bioreversible Carriers in Drug Design, edited by Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

The compositions described herein including Compound A may also include metabolites. When used herein, the term "metabolite" refers to a metabolite of Compound A or a pharmaceutically acceptable salt, analog or derivative thereof, which exhibits activity similar to the disclosed therapeutic agent in vitro or in vivo. The compositions described herein may also include hydrates and solvates. When used herein, the term "solvate" refers to a complex formed by a solute (therapeutic agent in this article) and a solvent. Such solvents for the purpose of the embodiments preferably should not adversely interfere with the biological activity of the solute. The solvent may be, for example, water, ethanol or acetic acid.

The compositions disclosed herein can stimulate an immune response in a subject (e.g., a mammalian subject). In various cases, the compositions stimulate iNKT cells in the subject. In various cases, the compositions stimulate an immune response as measured by an in vitro assay. In various cases, the compositions stimulate an immune response in the subject in vivo.

Liposomes and liposomal preparations

Described herein are liposomal formulations of iNKT modulators, such as liposomal formulations referred to herein as Compound A.

Liposomes are vesicles containing lipids, including one, two or several concentric lipid bilayers (usually composed of phospholipids), surrounding an aqueous core, which can be used to deliver hydrophilic and hydrophobic active pharmaceutical ingredients (API) to the body. Small hydrophilic active pharmaceutical ingredients can be encapsulated in the aqueous core (cavity) of the liposome, for example in a solution or suspension. Charged hydrophilic active pharmaceutical ingredients and uncharged hydrophobic active pharmaceutical ingredients can be bound to the membrane of the liposome by electrostatic or hydrophobic interactions or by covalent bonds, respectively. Liposomes can be, for example, by fusing liposomes with other bilayers (such as cell membranes), diffusing compounds out of liposomes, or digesting liposomes in macrophages to deliver active pharmaceutical ingredients to the body.

Many methods for preparing liposomes are known, generally involving applying some form of energy to a dispersion of phospholipids in a polar solvent. Exemplary methods include or involve ultrasound, extrusion, microfluidization, and heating. See, for example, Panahi et al., Artificial Cells, Naon medicine, and Biotechnology, 45: 4, 788-799 (2017), incorporated herein by reference. All liposome preparation methods are contemplated as embodiments for preparing the liposomes described herein.

As described in U.S. Patent No. 7,060,291, which is incorporated herein by reference, liposomes can have various sizes, for example, as low as 25nm or as high as 10,000nm or larger average diameter. Size is affected by many factors, such as lipid composition and preparation method, and is determined by many technologies, such as dynamic light scattering (DLS). Various methods can be used, such as ultrasound, homogenization, French Press application and grinding, to prepare larger liposomes into smaller sized liposomes. Extrusion (see, for example, U.S. Patent No. 5,008,050, incorporated herein by reference) can be used to produce liposomes with a predetermined average size by forcing liposomes under pressure to pass through a filter hole of a defined, selected size. Tangential flow filtration, as described in PCT Application No. WO 1989/008846, incorporated herein by reference, can also be used to adjust the size of liposomes (that is, to produce a liposome population with less size heterogeneity and more uniform, limited size distribution).

It is very advantageous to use liposomes to deliver active pharmaceutical ingredients (API) or multiple ingredients, such as IMM60, IMM70 or IMM80. Unlike micellar delivery vehicles, liposomes are physically stable. Liposome lipid compositions can be customized to deliver specific API to a targeted location, which limits the potential toxicity of ingredients. In addition, liposomes allow the delivery of a known amount of API because they minimize the loss of API, such as degradation, non-target organ removal and precipitation, such as detergents. Liposomes also provide a method for delivering adjuvants and antigens in a specific ratio, otherwise it is impossible to achieve by, for example, co-injection, as described in U.S. Patent Nos. 7,850,990 and 7,842,676, each of which is incorporated herein by reference.

In one aspect, described herein are compositions comprising liposomes, wherein the liposomes comprise Compound A or a salt, ester, solvate or hydrate thereof and two or more lipids, and a pharmaceutically acceptable diluent, excipient, adjuvant or carrier. In an embodiment, the liposomes comprise a lipid bilayer membrane surrounding an aqueous core, wherein the lipid bilayer membrane comprises two or more lipids (e.g., 2, 3, 4, 5, 6, 7 or 8 lipids).

Specifically contemplated are liposomes composed of Compound A and two or more lipids; exemplary lipid pairs for preparing liposomes include:

(a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG);

(b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol);

(c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB);

(d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC);

(e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); and

(f) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL).

Even when using API at low concentrations, these liposomes can provide the desired efficacy of API (e.g., IMM60). In experiments described herein, liposome delivery of IMM60 demonstrated efficacy in laboratory mouse models at various concentrations in the range of, for example, 0.01 ng/mL to 5000 ng/mL. As shown in Figures 1A to 1E, Figures 3 to 5, and Figures 7 to 8, as described herein, even if a small amount of API (e.g., 0.1 ng, 1 ng, and 5 ng) is delivered (e.g., intravenously) in liposome form, excellent therapeutic effects measurable by many relevant biomarkers can be provided, while only delivering dissolved API in such a low amount does not provide the same level of efficacy. As shown in Figures 6 to 8, a 0.1 ng IMM60 dose formulated in liposomes produced a superior immune response (measured by multiple markers in preclinical rodent models) compared to a formulation without liposomes.

In certain embodiments, the liposome in the composition has an average diameter less than about 200nm. In certain embodiments, the average diameter of the liposome in the composition is about 50nm to about 200nm, or about 50nm to about 170nm, or about 70nm to about 130nm, or about 60nm to about 160nm, or about 70nm to about 150nm, or about 75nm to about 145nm, or about 90nm to about 130nm, or about 75nm to about 145nm. In certain embodiments, the average diameter of the liposome in the composition is about 50nm, about 55nm, about 60nm, about 65nm, about 70nm, about 75nm, about 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm or about 150nm. In an embodiment, the average diameter standard deviation of the liposomes in the composition can be about 40 nm, for example, the average liposome diameter of the composition is about 100 nm ± 40 nm or about 110 nm ± 40 nm. As used herein, the average diameter of the liposomes in the composition is determined by dynamic light scattering (DLS).

All integer size ranges and integer subranges within the size range of 50 nm to 200 nm are specifically contemplated herein as exemplary average diameters of liposomes in the compositions of the invention. To assess particle size, size measurements were performed using 90° dynamic light scattering.

In some embodiments, the average diameter of the liposomes in the composition is within a size range, the lower end of the size range being any size selected from the group consisting of about 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm; and the upper end of the size range is any size selected from the group consisting of about 200 nm, 190 nm, 175 nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145nm, 144nm, 143nm, 142nm, 141nm, 140nm, 139nm, 138nm, 137nm, 136nm, 1 35nm, 134nm, 133nm, 132nm, 131nm, 130nm, 129nm, 128nm, 127nm, 126nm, 125 nm, 124nm, 123nm, 122nm, 121nm, 120nm, 119nm, 1 18nm, 117nm, 116nm, 115nm, 114nm, 113nm, 112nm, 111nm, 110nm, 109nm, 108nm, 107nm, 106nm, 105nm, 104nm, 103nm, 102nm, 101nm, 100nm, 99nm, 98nm, 97nm, 96nm, 95nm, 94nm, 93nm, 92nm, 91nm or 90nm.

In an embodiment, the average diameter of the liposome in the composition is in the range of about 50 nm to about 200 nm. In an embodiment, the average diameter of the liposome in the composition is in the range of about 50 nm to 170 nm, or about 70 nm to about 150 nm, or about 90 nm to about 130 nm, or about 110 nm ± 60 nm, or about 110 nm ± 40 nm, or about 110 nm ± 20 nm.

In some embodiments, the composition exhibits a degree of uniformity in liposome size/mass in the formulation, which can be expressed by measurement (or derivation) of the polydispersity index (PdI). PdI is defined as the standard deviation of the particle diameter distribution divided by the square of the mean particle diameter, as shown in the following formula, where "a" is the radius of the particle:

PdI is used to estimate the uniformity of particle distribution, and a larger PdI value corresponds to a larger particle size distribution in the sample particles. For example, in some embodiments, the PdI of the composition is less than or equal to about 0.4, or less than or equal to about 0.35, or less than or equal to about 0.3, or less than or equal to about 0.25, or less than or equal to about 0.23, or less than or equal to about 0.22, or less than or equal to about 0.21, or less than or equal to about 0.20, or less than or equal to about 0.19, or less than or equal to about 0.18, or less than or equal to about 0.17, or less than or equal to about 0.16, or less than or equal to about 0.15, or less than or equal to about 0.14, or less than or equal to about 0.13, or less than or equal to about 0.12, or less than or equal to about 0.11, or less than or equal to about 0.10. In some variations, the PdI is in the range of 0.01-0.40; or in the range of 0.02-0.35; or in the range of 0.03-0.30; or in the range of 0.04-0.25; or in the range of 0.05-0.20; or in the range of 0.0-0.15.

To calculate PdI herein, the particle diameter (or radius) was obtained using particle size measurements performed using 90° dynamic light scattering.

Compound A can be present in liposomes in any amount, which provides one or more of the following: liposomes with similar efficacy to corresponding soluble Compound A, which Compound A is not incorporated into liposomes or provides excellent efficacy; or providing a therapeutic window (between the minimum effective concentration and the toxic concentration). In some embodiments, based on the gross weight of the liposome, Compound A is present in the liposome in an amount of about 0.1% by weight to about 20% by weight. In some embodiments, based on the gross weight of the liposome, Compound A is present in an amount of about 0.5% by weight to about 20% by weight, or about 3% by weight to about 8% by weight, or about 4% by weight to about 13% by weight. For example, based on the gross weight of the liposome, Compound A can be present in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%. In an embodiment, based on the gross weight of the liposome, Compound A is present in an amount of about 0.5 wt% to about 15 wt%, or about 1 wt% to about 12 wt%, or about 5 wt% to about 12 wt%. The weight % of Compound A is selected to produce an effective immunomodulatory response with minimal toxic and side effects. If too little Compound A is present relative to total liposomes, liposomes will not stimulate a sufficiently strong reaction. If there is too much Compound A relative to total liposomes, excessive Compound A will compete for binding sites, and may cause liposomes to block binding to each other. Furthermore, too much Compound A may cause toxicity problems, for example, where Compound A is present in an amount exceeding 20 wt % based on the total weight of the liposome.

In estimating the weight % of liposome components, the weight of the liposome membrane is taken into account, ie, the ingredients used to prepare the liposome membrane, and the weight of the aqueous or hydrophilic lumen of the liposome is not taken into account.

In some embodiments, the liposome composition is provided in the form of a concentrated formulation. For example, consider a formulation comprising Compound A at a concentration of 0.01 mg/ml to 50 mg/ml. Within this range, all subranges are contemplated. For example, subranges with the following exemplary low concentration limits are contemplated: 0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.075 mg/ml, 0.1 mg/ml, 0.125 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.25 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.75 mg/ml, and 1.0 mg/ml; and subranges with the following exemplary high concentration limits (higher than the selected low concentration limits) are contemplated: 0.5 mg/ml, 0.75 mg/ml, 0 mg/ml, 1.25 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 5.0 mg/ml, 10 mg/ml, 5 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, and 40 mg/ml. Concentrated formulations can be used for preparation, storage, and distribution.

In some embodiments, it is contemplated to use a liposome composition at a lower concentration. For example, in some embodiments, for administration, a low concentration composition is prepared by diluting a concentrated formulation with a pharmaceutically acceptable diluent, such as an isotonic saline solution. In some embodiments, the composition comprises a concentration of about 0.001 ng/mL to about 10,000 ng/mL of Compound A. For example, it is contemplated to include a composition of Compound A at a concentration of about 0.01 ng/mL to about 5000 ng/mL, or about 0.1 ng/mL to about 5000 ng/mL, or about 1 ng/mL to about 1000 ng/mL, or about 10 ng/mL to about 100 ng/mL. Exemplary concentrations of Compound A contemplated include 0.01 ng/mL, 0.5 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 0.75 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 0.75 ng/mL, 1 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 7.5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1 μg/mL, 2 μg/mL, 2.5 μg/mL, 5 μg/mL, 7.5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 75 μg/ml, and 100 μg/ml. Data from preclinical mouse studies showed that concentrations as low as 0.01 ng/mL were effective in mice injected with 100 uL into the tail.

Compound A described herein can be synthesized using any means known to a synthetic organic chemist, such as the synthetic methods described in U.S. Pat. No. 9,700,532, which is incorporated herein by reference.

In some embodiments, the two or more lipids disclosed herein may comprise a lipid containing a saturated distearoyl moiety and a lipid containing a phosphoglycerol moiety.

For example, embodiments are contemplated in which at least one lipid is selected from the group consisting of phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidyl Serine (DOPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylethanolamine (DOPE), palmitoleoylphosphatidylcholine (POPC), palmitoleoylphosphatidylserine (POPS), palmitoleoylphosphatidylethanolamine (POPE), diacylphosphatidylethanolamine 4-(N-maleimidomethyl-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and combinations thereof.

In an embodiment, the lipid comprising a saturated distearoyl moiety may further comprise a phosphorylcholine moiety, a phosphoglycerol moiety, a phosphate moiety, a phosphoethanolamine moiety, a phosphoacetamide moiety, a polyethylene glycol moiety, and the like.

In some embodiments, sterols such as cholesterol, ergosterol, lanosterol, and combinations thereof are contemplated.

In an embodiment, the lipid comprising a phosphoglycerol moiety can further comprise a saturated distearoyl moiety, a dimyristoyl moiety or a palmitoyl moiety. For example, two or more lipids disclosed herein can comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG).

In some embodiments, the two or more lipids disclosed herein may comprise any of the following lipid pairs:

(a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG);

(b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol);

(c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB);

(d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC);

(e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); or

(f) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL).

In some embodiments, the two or more lipids include 2-oleoyl-1-palmitoylglycerol-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB). In some embodiments, the liposomes include two lipids, the lipids including 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG). In embodiments, the two or more lipids may include (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG); (b) 2-oleoyl-1-palmitoylglycerol-3-phosphocholine (POPC) and 3β-[N-

(N', N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol); or (c) 2-oleoyl-1-palmitin glycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB). In an embodiment, the two or more lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG). In an embodiment, the two or more lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG) and cholester-5-ene-3β-ol (CHOL).

In an embodiment, the two or more lipids included in the liposome may further include any lipid capable of forming a liposome with Compound A as described herein, as long as the resulting liposome has an effect that is at least similar to or enhanced with the corresponding soluble Compound A not incorporated into the liposome. For example, one or more lipids as described in U.S. Patent Application Publication No. 2014/0050780A1 may be included in the liposome, and the patent application disclosure is incorporated herein by reference. In an embodiment, the liposome may include three lipids. In an embodiment, the liposome may include four lipids. Each of the two or more lipids may independently have a net positive charge (i.e., a cationic lipid), a net negative charge (i.e., an anionic lipid), no charge (i.e., a nonionic lipid), or an equal number of positive and negative charges (i.e., zwitterionic lipids). Lipids may include those lipids that form a bilayer by themselves or with Compound A, and those lipids that do not form a bilayer alone, but they may be part of a stable bilayer made of one or more other lipids.

In embodiments, additionally, any lipid described herein is contemplated to be a salt thereof, e.g., an ammonium salt, a sodium salt, a sodium/ammonium salt, a lithium salt, a potassium salt, etc.

Based on the gross weight of the liposome, the amount of lipid that can be present in the liposome is about 75 wt % to about 99.9 wt %, or about 80 wt % to about 98 wt %, or about 80 wt % to about 95 wt %, or about 85 wt % to about 95 wt %. In certain embodiments, based on the gross weight of the liposome, lipid is present in an amount of about 75 wt % to about 99 wt %, or about 75 wt % to about 98 wt %, or about 75 wt % to about 95 wt %, or about 87 wt % to about 93 wt %. For example, the lipid can be present in an amount of 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% by weight, based on the total weight of the liposome.

The mass ratio of Compound A to the two or more lipids can be about 1:5 to about 1:20, or about 1:7 to about 1:15, or about 1:7 to about 1:12. In some embodiments, the mass ratio of Compound A to the two or more lipids can be about 1:8 to about 1:10, or about 1:9. For example, the mass ratio of Compound A to the two or more lipids can be about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20.

In some embodiments, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG), the DSPC is present in an amount of about 40% to 80% by weight, or about 40% to about 60% by weight, or about 45% to about 55% by weight, based on the total weight of the liposome. For example, DSPC is present in an amount of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, based on the total weight of the liposome.

In embodiments, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG), the DSPG is present in an amount of about 20% to 60% by weight, or about 30% to about 50% by weight, or about 35% to about 45% by weight, based on the total weight of the liposome. For example, DSPG is present in an amount of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% by weight, based on the total weight of the liposome.

In certain embodiments, two or more lipids further comprise CHOL.In an embodiment, two or more lipids comprise DSPC, DSPG and CHOL, wherein, CHOL exists with the amount of about 1 % by weight to about 20 % by weight.For example, CHOL can exist with the amount of about 5 % by weight to about 15 % by weight, or about 8 % by weight to about 12 % by weight, such as about 10 % by weight.In an embodiment, two or more lipids comprise DSPC, DSPG and CHOL, wherein DSPC exists with the amount of about 40 % by weight to about 50 % by weight, DSPG exists with the amount of about 30 % by weight to about 40 % by weight, and CHOL exists with the amount of about 5 % by weight to about 15 % by weight, for example DSPC exists with the amount of about 45 % by weight, DSPG exists with the amount of about 35 % by weight, and CHOL exists with the amount of about 10 % by weight.

In an embodiment, two or more lipids include 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol). In an embodiment, POPC is present in an amount of about 40% to 80% by weight, or about 45% to about 65% by weight, or about 50% to about 60% by weight, based on the gross weight of the liposome. In an embodiment, DC-Chol is present in an amount of about 15% to 55% by weight, or about 20% to about 50% by weight, or about 25% to about 35% by weight, based on the gross weight of the liposome.

In an embodiment, two or more lipids comprise 2-oleoyl-1-palmitin glycerol-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB). In an embodiment, based on the gross weight of this liposome, POPC is present in an amount of about 40 wt % to 80 wt %, or about 45 wt % to about 65 wt %, or about 50 wt % to about 60 wt %. In an embodiment, based on the gross weight of this liposome, DC-Chol is present in an amount of about 15 wt % to 55 wt %, or about 20 wt % to about 50 wt %, or about 30 wt % to about 40 wt %.

In an embodiment, two or more lipids include L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC). In an embodiment, EPG is present in an amount of about 40% to 80% by weight, or about 55% to about 75% by weight, or about 60% to about 70% by weight, based on the gross weight of the liposome. In an embodiment, EPC is present in an amount of about 10% to 50% by weight, or about 15% to about 40% by weight, or about 15% to about 30% by weight, based on the gross weight of the liposome.

In an embodiment, two or more lipids include 2-oleoyl-1-palmitin glycerol-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In an embodiment, based on the gross weight of the liposome, POPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 65 wt %, or about 45 wt % to about 55 wt %. In an embodiment, based on the gross weight of the liposome, DOTAP is present in an amount of about 20 wt % to 50 wt %, or about 25 wt % to about 45 wt %, or about 35 wt % to about 45 wt %.

In an embodiment, two or more lipids include 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-ene-3β-ol (CHOL). In an embodiment, DMPG is present in an amount of about 40% to 80% by weight, or about 50% to about 75% by weight, or about 60% to about 70% by weight, based on the gross weight of the liposome. In an embodiment, CHOL is present in an amount of about 10% to 50% by weight, or about 15% to about 35% by weight, or about 15% to about 30% by weight, based on the gross weight of the liposome.

In an embodiment, the liposome further comprises a liposome buffer. For example, in an embodiment, the liposome buffer may comprise phosphate, sodium chloride, sucrose or a combination thereof. In an embodiment, the liposome buffer comprises sodium phosphate and sodium chloride. In an embodiment, the liposome buffer comprises sodium chloride and sucrose. In an embodiment, the liposome buffer comprises sodium chloride, phosphate and sucrose. In an embodiment, the pH of the liposome buffer is about 1% to 7, or about 6.3 to about 6.7, such as 6.5.

Antigen-carrying liposomes

In some embodiments, the liposomes described herein comprise at least one antigen, such as a viral antigen, a bacterial antigen, a fungal antigen, a tumor antigen, or a mixture thereof. The exact amount of the antigen relative to the liposome depends on the composition and purpose of the antigen and can be determined by a person skilled in the art. In these embodiments, the liposomes described herein can be used as an immunostimulant or adjuvant to produce a protective immune response to the administered antigen, such as a B cell response, an IgG antibody response, a T cell response, or a CTL response.

The antigen can be any antigen material suitable for treating a particular disease. In some embodiments, the liposomes described herein contain antigens and are used to treat cancer. The antigen can be a full-length protein antigen, a long peptide antigen (i.e., a peptide containing at least 25 amino acids, such as 27-75, 25-50, 25-40 or 25-30 amino acids), or a short peptide antigen (i.e., a polypeptide containing 6-25 amino acids, such as 6-25, 8-25, 10-25 or 15-20 amino acids). In these embodiments, the antigen can be a tumor-associated peptide or protein that induces or enhances an immune response and is derived from tumor-associated genes and encoded proteins, including, for example, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(AGE-B4), tyrosinase, brain glycogen phosphorylase, melan-A, MAGE-C1, MAGE-C2、NY-ES O-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, α-lectin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, H LA-A11, hsp70-2, KIAAO205, Mart2, Mum-2 and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triphosphate isomerase, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, /Lage-2, SP17 and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO(LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TAG72, TLP and TPS. For example, the antigenic peptides with tumor characteristics include those listed in International Patent Application Publication No. WO 20000/020581 and U.S. Patent Application Publication No. 2010/0284965, each of which is incorporated herein by reference. In some embodiments, the antigen is a tumor antigen selected from the group consisting of: MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, and mixtures thereof. In some embodiments, the tumor antigen is selected from the group consisting of: P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1 , CAGE, LB33/MUM-1, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE and mixtures thereof. In some embodiments, the tumor antigen comprises NY-ESO-1 or an antigenic fragment thereof. In some variants, the tumor antigen is a mammalian protein. In some variants, the tumor antigen is a human protein. In some variants, the full-length protein is used as an antigen. In some variants, peptides comprising antigenic fragments of these proteins are used as tumor antigens. In some variants, antigenic fragments of these antigens are used as tumor antigens.

Small hydrophilic antigens can be encapsulated within the core of the liposome. Small or large hydrophobic antigens can be non-covalently bound to the non-polar portion of the lipid bilayer (e.g., by hydrophobic interactions), charged small or large antigens can be attached (e.g., by electrostatic interactions) to charged portions of the exterior of the lipid bilayer, and small or large hydrophobic or hydrophilic antigens can also be covalently linked to any portion of the liposome membrane.

In the embodiments described herein where the antigen is carried by liposomes, the lipids used to form the liposomes are determined by the size and charge of the antigen (eg, a small, hydrophilic, hydrophobic, positively charged, or negatively charged antigen).

In embodiments where the positively charged antigen is non-covalently associated with the liposome membrane, at least one of the two or more lipids preferably has a net negative charge and is selected from the group consisting of anionic sphingosines (e.g., dimethylsphingosine-1-phosphate, ceramide phosphate, dihydroceramide phosphate, gangliosides, and sulfates), anionic phospholipids (e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, inositol phosphates, cardiolipin, bis(monoacylglycerol) phosphates, anionic detergents that are not sphingolipids or phospholipids, and anionic bioactive lipids (e.g., adjuvants, liponucleotides, TLR-4 agonists, and diacylglycerol pyrophosphate).

In embodiments where the negatively charged antigen is non-covalently associated with the liposome membrane, at least one of the two or more lipids preferably has a net positive charge. In these embodiments, the cationic lipid can be a cationic sphingosine (e.g., trimethylsphingosine, trimethylphytosphingosine, and pyridinium ceramide), a cationic lipid that is not a sphingosine (e.g., 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (18:1 TAP, DOTAP), 1,2-dioleoyl-3-trimethylammonium-propane (methylsulfate salt) (18:1 TAP, DOTAP, MS salt), 1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt) (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (chloride salt) (18:0TAP), transfection reagent I (i.e., containing 1:1 w/w ratio of DOTAP:DOPE), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) (18:1DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0DAP), dimethyldioctadecyl ammonium (bromide salt) (18:0DDAB), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt) (12:0EPC, Cl salt), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (14:0EPC, Cl salt), 1,2-Dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt) (14:1EPC, Tf salt), 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (16:0EPC, Cl salt), 1,2-Distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:0EPC, Cl salt), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:1EPC, Cl salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (16:0-18:1EPC, Cl salt) and cationic bioactive lipids (e.g., dimethyldioctadecylammonium (bromide salt) (18:0DDAB), lysylphosphatidylglycerol).

Lipids that can be used to prepare liposomes (having a bilayer to which a hydrophobic antigen can be non-covalently bound) include, but are not limited to, phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidic acid (PA).

Therapeutic agents

In some embodiments, the liposome further comprises at least one therapeutic agent in the core of the liposome. In some embodiments, the at least one therapeutic agent is selected from the group consisting of an immunomodulator, a Toll-like receptor agonist, a Nod ligand, an antiviral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immunotherapeutic agent, a chemotherapeutic agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and a combination thereof.

In some embodiments, the composition further comprises at least one therapeutic agent. In some embodiments, the at least one therapeutic agent is selected from the group consisting of an immunomodulator, a Toll-like receptor agonist, a Nod ligand, an antiviral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immunotherapeutic agent, a chemotherapeutic agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and a combination thereof.

Composition and route of administration

As described herein, the compositions described herein comprise a pharmaceutically acceptable excipient, diluent, adjuvant, or carrier. The compositions comprising Compound A are administered by any route that permits treatment of the disease or condition.

Any standard route of administration can be used, including parenteral administration, such as intravenous (IV), intraarterial (IA), perivascular, intraperitoneal (IP), intrapulmonary, subcutaneous (SC) or intramuscular (IM), intrathecal, topical, transdermal, rectal, oral, nasal or by inhalation to deliver the composition comprising Compound A to the patient. In some embodiments, the composition disclosed herein is administered to the patient intravenously or intramuscularly. In an embodiment, the composition is administered intravenously. Sustained-release formulations can also be prepared from the reagents described herein to achieve controlled release of the active agent (Compound A) in contact with body fluids in the gastrointestinal tract, and provide a substantially constant and effective content level of the active agent in the plasma.

In cancer patients, intratumoral injection is particularly contemplated as an embodiment.

Administration can take the form of a single dose administration, or the compositions disclosed herein can be administered in divided doses over a period of time or in a continuous release formulation or administration method (e.g., a pump). However, the composition described in the embodiments is selected to be administered to a subject, the amount of the composition administered, and the route of administration selected to permit effective treatment of the disease condition.

In an embodiment, the composition is formulated with one or more pharmaceutically acceptable excipients, carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the specific mode of administration and dosage form. Depending on the formulation and route of administration, the composition should generally be formulated to achieve a physiologically compatible pH, and may be in the range of about pH 3 to about pH 11, preferably in the range of about pH 3 to about pH 7. In an alternative embodiment, the pH value is adjusted to a range of about pH 5.0 to about pH 8. In an embodiment, the composition may include a therapeutically or prophylactically effective amount of Compound A as described herein and one or more pharmaceutically acceptable excipients. Optionally, the composition may include a combination of compounds described herein, or may include a second active ingredient (e.g., an antibacterial or antimicrobial agent) that can be used to treat or prevent bacterial infections.

Disclosed herein, for example, compositions for parenteral or oral administration are generally solid, liquid solution, suspension, and inhalable formulations for pulmonary administration are generally liquid or powder. Compositions disclosed herein can also be formulated as lyophilized solids, which are reconstituted with physiologically compatible solvents before administration. Lyophilization (freeze drying) removes moisture by sublimation at low temperature and low pressure, and preserves sensitive materials such as drugs, vaccines and probiotic cultures. Compositions disclosed herein can be formulated as solutions, which are reconstituted with physiologically compatible solvents before administration for intravenous or intramuscular injection. Parenteral injection or extended infusion, for example 1, 2, 3, 4, 5, 6, 7, 8, 12 or 24 hours, can be performed.

In some embodiments, the compositions described herein comprise lipid bilayer-coated particles instead of or in addition to liposomes.

The term "pharmaceutically acceptable excipient" refers to an excipient used in the administration of a pharmaceutical agent, such as a compound described herein. The term refers to any pharmaceutical excipient that can be administered without undue toxicity.

Pharmaceutically acceptable excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions (see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients can be carrier molecules, including large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inert viral particles. Other excipients include antioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA), carbohydrates (e.g., dextrin, hydroxyalkyl cellulose and/or hydroxyalkyl methyl cellulose), stearic acid, liquids (e.g., oil, water, saline, sodium phosphate, including sodium phosphate monohydrate, glycerol and/or ethanol) wetting agents or emulsifiers, pH buffer substances, etc.

In another embodiment, the compositions disclosed herein can be formulated as a suspension comprising Compound A and two or more lipids of the embodiments mixed with at least one pharmaceutically acceptable excipient suitable for preparing a suspension.

The compositions disclosed herein may also be in the form of a solution of a salt form of the active ingredient in a suitable aqueous vehicle (such as water or isotonic saline solution or dextrose solution). Also contemplated are compounds that have been modified by substitution or addition of chemical or biochemical moieties, such as by PEGylation to make the compound more suitable for delivery (e.g., to increase solubility, biological activity, palatability, reduce adverse reactions, etc.).

In some embodiments, the compounds described herein may be formulated for oral administration in the form of lipid-based formulations suitable for poorly solubility compounds.

An adjuvant is a substance that is incorporated into or administered with an antigen that enhances the immune response. An adjuvant can enhance the immune response by providing a reservoir of antigen (extracellular or intracellular), activating macrophages, and stimulating specific groups of lymphocytes. Many adjuvants are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a homologue obtained by purification and acid hydrolysis of Salmonella Minnesota RS 595 lipopolysaccharide; saponins, including QS21 (SmithKline Beecham), pure QA-21 saponin purified from a Quillaja tree extract; DQS21 (SmithKline Beecham) described in PCT application WO 96/33739; QS-7, QS-17, QS-18 and QS-L1 (So et al., Mol Cells (1997) 7:178-186); ISCOMATORIX adjuvant, a cage-like structure composed of saponins, phospholipids and cholesterol (see, e.g., Maraskovsky et al., Clin. Cancer Res. (2004) 10:2879-2890); incomplete Freund's adjuvant; complete Freund's adjuvant; montanide; alum; CpG oligonucleotides (see, e.g., Kreig et al., Nature 374:546-9, 1995); and other immunostimulatory oligonucleotides, including polyIC and polyICLC ; and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol.

Treatment

Provided herein is a method for treating different types of cancers in a subject (e.g., a mammal) in need thereof, the method comprising administering a composition as described herein to the subject in a therapeutically effective amount. In some embodiments, the subject is a mammalian subject. In some cases, the mammalian subject is a human subject. It is also contemplated that the methods described herein are practiced in other mammalian subjects, and in particular, it is also contemplated that the practice is routinely used as a model for demonstrating therapeutic efficacy in humans (e.g., primates, pigs, dogs, or rabbits). It is expected that the mice used in preclinical studies may be 1000 times or more sensitive to Compound A than humans, which provides guidance for adjusting dosing. Standard dose response studies are used to optimize dosage and dosing regimens.

Provided herein is the use of a composition disclosed herein in the preparation of a medicament, such as the medicament described above, for treating cancer in a mammalian subject.

The disclosed methods can be used to treat cancer, for example, to inhibit cancer growth, including complete remission of cancer, to inhibit cancer metastasis, and to promote cancer resistance. The term "cancer growth" generally refers to any of a number of indicators that indicate a change in a cancer to a more advanced form. Thus, indicators for measuring inhibition of cancer development include, but are not limited to, a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasonography, or other imaging methods), delayed tumor growth, destruction of tumor blood vessels, improved performance in delayed hypersensitivity skin tests, an increase in cytolytic T lymphocyte activity, and a decrease in tumor-specific antigen levels.

The term "cancer resistance" refers to an increased ability of a subject to resist the growth of cancer, particularly the growth of an existing cancer. In other words, the term "cancer resistance" refers to a decreased propensity for cancer growth in a subject.

On the one hand, cancer can include solid tumors, such as epithelial cancer or sarcoma. Carcinomas include malignant neoplasms derived from epithelial cells that infiltrate (e.g., invade) surrounding tissues and cause metastasis. Adenocarcinomas are carcinomas derived from glandular tissue or derived from tissues that form recognizable glandular structures. Another major class of cancers includes sarcomas and fibrosarcomas, which are tumors in which cells are embedded in fibrous or uniform substances (such as embryonic connective tissue). The present invention also provides methods for treating cancers of the bone marrow or lymphatic system, including leukemias, lymphomas, and other cancers that typically do not exist as tumor masses but are distributed in blood vessels or lymphatic reticulum. Further consideration is given to methods for treating the following: adult and pediatric tumors, growth of solid tumors/malignant tumors, myxoid and round cell carcinomas, locally advanced tumors, cancer metastasis, including lymphatic metastasis. The cancers listed herein are not intended to be limited. Age (children and adults), sex (males and females), primary and secondary, before and after metastasis, acute and chronic, benign and malignant, anatomical location cancer embodiments and variations are all targets that can be considered. Cancers are grouped by embryonic origin (eg, carcinoma, lymphoma, and sarcoma), by organ or physiological system, and by miscellaneous groupings. Specific cancers may overlap in their classification, and their listing in one group does not exclude them from another group.

Epithelial cancers that can be targeted include the following cancers and carcinomas: adrenocortical cancer, acinar cancer, acinic cell cancer, acinous cancer, adenocystic cancer, adenoid cystic cancer, adenoid squamous cell cancer, canceradenomatosum, adenosquamous cancer, adnexal cancer, cancer of the adrenal cortex, adrenocortical cancer, aldosterone-producing cancer, aldosterone-secreting cancer, alveolar cancer, alveolar cell cancer, ameloblastic cancer, ampullary cancer, anaplastic cancer of thyroid gland, apocrine cancer, basal cell cancer, basal cell cancer, alveolar cancer, comedo basal cell cancer, ulcerative colitis, ... cell carcinoma, duct carcinoma, ductal carcinoma, ductal carcinoma of the prostate, ductal carcinoma in situ (DCIS), eccrine carcinoma, embryonal carcinoma, cancer encuirasse, endometrial carcinoma, cancer of endometrium, endometrioid carcinoma, epidermoid tumor, cancer ex mixed tumour, cancer ex pleomorphic adenoma, exophytic carcinoma, fibrolamellar carcinoma, cancer fibrosum, follicular thyroid carcinoma, gastric carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma of the thyroid, cancer gigantocellulare, glandular carcinoma, granular cell carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, adrenal tumor, infantile embryonal carcinoma embryonal carcinoma, islet cell carcinoma, inflammatory breast cancer, carcinoma in situ, intraductal carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, juvenile embryonal carcinoma, Kulchitsky cell carcinoma, large cell carcinoma, leptomeningeal carcinoma, lobular carcinoma, invasive lobular carcinoma, invasive lobular carcinoma, lobular carcinoma in situ (LCIS), lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, medullary cancer of thyroid gland, medullary thyroid cancer, melanoma, meningeal carcinoma, Merkel cell carcinoma, metatypical cell carcinoma, micropapillary carcinoma, mucinous carcinoma, cancer muciparum, cancer mucocellulare, mucoepidermoid carcinoma, mucinous carcinoma mucosum), mucous carcinoma, nasopharyngeal carcinoma, neuroendocrine carcinoma of the skin, non-invasive carcinoma, non-small cell carcinoma, non-small cell lung cancer (NSCLC), oat cell carcinoma, cancer ossificans, osteoid carcinoma, Paget's carcinoma, papillary carcinoma, papillary carcinoma of the thyroid, periampullary carcinoma, preinvasive carcinoma, acanthate cell carcinoma, primary intraosseous carcinoma, renal carcinoma, scar carcinoma, Schistosoma bladder carcinoma, Schneiderian carcinoma, scirrhous carcinoma, sebaceous carcinoma, signet ring cell carcinoma, cancer simplex, small cell carcinoma, small cell lung cancer (SCLC), spindle cell carcinoma, cavernous carcinoma, squamous carcinoma, squamous cell carcinoma, terminal duct carcinoma, anaplastic thyroid thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, papillary thyroid cancer, trabecular cancer of the skin, transitional cell cancer, tubular cancer, undifferentiated cancer of thyroid gland, cancer of the uterine corpus, verrucous carcinoma, villous carcinoma, cancer villosum, yolk sac cancer, squamous cell carcinoma, especially of the head and neck, squamous cell carcinoma of the esophagus, and oral cancers and tumors.

Sarcomas that may be targeted include liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, avian sarcoma, botryoid sarcoma, sarcoma botryoides, chicken sarcoma, chloromatous sarcoma, chondroblastic sarcoma, clear cell sarcoma of the kidney, embryonal sarcoma, endometrial stromal sarcoma, epithelioid sarcoma, Ewing sarcoma, fascial sarcoma, fibroblastic sarcoma, fowl sarcoma, giant cell sarcoma, granulocytic sarcoma, hemangioendothelial sarcoma, Hodgkin sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen sarcoma, Kaposi sarcoma, Kupffer cell sarcoma, sarcoma, sarcoma of soft tissue, spindle cell sarcoma, synovial sarcoma, sarcoma of telangiectatic type, sarcoma of osteosarcoma/malignant fibrous histiocytoma of bone, and sarcoma of soft tissue.

Lymphomas that can be targeted include AIDS-related lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, T-cell leukemia/lymphoma, African lymphoma, B-cell lymphoma, B-cell monocytoid lymphoma, bovine malignant lymphoma, Burkitt's lymphoma, centrocytic lymphoma, cutaneous lymphoma, diffuse lymphoma, diffuse large cell (diffuse, large cell) lymphoma, diffuse mixed small and large cell (diffuse, mixed small and large cell) lymphoma, diffuse small cleaved cell (diffuse, smallcleaved cell) lymphoma, follicular lymphoma, follicular centrocytic lymphoma, follicular, mixed small cleaved and large cell (follicular, mixed small cleaved and large cell) lymphoma, follicular, predominantly large cell (follicular, predominantly large cell) lymphoma, and follicular, mixed small cleaved and large cell lymphoma. cell lymphoma, follicular, predominantly small cleaved cell lymphoma, giant follicle lymphoma, giant follicular lymphoma, granulomatous lymphoma, histiocytic lymphoma, large cell lymphoma, immunoblastic lymphoma, large cleaved cell lymphoma, large non-cleaved cell lymphoma, Lennert lymphoma, lymphoblastic lymphoma, intermediate lymphoma; intermediately differentiated lymphoma, plasmacytoid lymphoma; poorly differentiated lymphocytic lymphoma, small lymphocytic lymphoma, well differentiated lymphocytic lymphoma lymphocytic) lymphoma, bovine lymphoma; MALT lymphoma, mantle cell lymphoma, mantle zone lymphoma, marginal zone lymphoma, Mediterranean lymphoma, mixed lymphocyte-histiocytic lymphoma, nodular lymphoma, plasmacytoid lymphoma, pleomorphic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, small B-cell lymphoma, small cleaved cell lymphoma, small non-cleaved cell lymphoma, T-cell lymphoma; convoluted T cell lymphoma, cutaneous T-cell lymphoma, small lymphocytic T-cell lymphoma, undefined lymphoma, undifferentiated U-cell (u-cell, undifferentiated) lymphoma, AIDS-related lymphoma, central nervous system lymphoma, cutaneous T-cell lymphoma, effusion (body cavity-based) lymphoma, thymic lymphoma, and cutaneous T-cell lymphoma.

Leukemias and other blood cell malignancies that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, erythroleukemia, lymphoblastic leukemia, myeloid leukemia, lymphocytic leukemia, myeloid leukemia, leukemia, hairy cell leukemia, T-cell leukemia, monocytic leukemia, myeloblastic leukemia, granulocytic leukemia, gross leukemia, hand mirror cell (hand mirror-cell leukemia, basophilic leukemia, hemoblastic leukemia, histiocytic leukemia, leukopenic leukemia, lymphatic leukemia, Schilling's leukemia, stem cell leukemia, myelomonocytic leukemia, monocytic leukemia, prolymphocytic leukemia, promyelocytic leukemia, micromyeloblastic leukemia, megakaryoblastic leukemia ) leukemia, megakaryocytic, Riedel cell, bovine leukemia, aleukemic leukemia, mast cell leukemia, myelocytic leukemia, plasma cell leukemia, subleukemia, multiple myeloma, nonlymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, myelodysplasia, and chronic myelocytic leukemia.

Brain and central nervous system (CNS) cancers and tumors that may be targeted include astrocytomas (including cerebellar astrocytomas and cerebral astrocytomas), brain stem gliomas, brain tumors, malignant gliomas, ependymomas, glioblastomas, medulloblastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, primary CNS lymphomas, ependymomas, brain stem gliomas, visual pathway and hypothalamic gliomas, extracranial germ cell tumors, medulloblastomas, myelodysplastic syndromes, oligodendrocytomas, myelodysplastic/myeloproliferative disorders, myeloid leukemias, myeloid leukemias, multiple myeloma, myeloproliferative disorders, neuroblastomas, plasma cell neoplasms/multiple myeloma, CNS lymphomas, intrinsic brain tumors, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion in the CNS.

Gastrointestinal cancers that can be targeted include extrahepatic bile duct cancer, colon cancer, colon and rectal cancer, colorectal cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, bladder cancer, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer, rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia.

Lung and respiratory cancers that can be targeted include bronchial adenoma/carcinoid, esophageal cancer, esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumors, non-small cell lung cancer, small cell lung cancer, small cell lung cancer, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer; paranasal sinus and nasal cavity cancer and pleuropulmonary blastoma.

Urinary tract and reproductive cancers that may be targeted include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, spleen cancer, kidney cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, penile cancer, renal cell carcinoma (including epithelial carcinoma), renal cell carcinoma, cancer of the renal pelvis and ureter (transitional cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, testicular cancer, cancer of the ureter and renal pelvis, transitional cell carcinoma, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian cancer, primary peritoneal epithelial neoplasms, cervical cancer, uterine cancer and solid tumors in ovarian follicles, superficial bladder tumors, invasive bladder transitional cell carcinoma, and muscle invasive bladder cancer.

Skin cancers and melanomas (and non-melanomas) that can be targeted include cutaneous T-cell lymphoma, intraocular melanoma, tumor progression of human skin keratinocytes, basal cell carcinoma, and squamous cell carcinoma. Liver cancers that can be targeted include extrahepatic bile duct carcinoma and hepatocellular carcinoma. Eye cancers that can be targeted include intraocular melanoma, retinoblastoma, and intraocular melanoma. Hormonal cancers that can be targeted include: parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumors, pituitary tumors, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, adrenocortical carcinoma, and ACTH-producing tumors.

Various other cancers that can be targeted include advanced cancer, AIDS-related cancer, anal cancer, adrenocortical cancer, aplastic anemia, aniline-induced cancer (aniline), betel-induced cancer (betel), buyocheek, medullary cancer, chimney-sweeps, clay pipe, colloid cancer, contact cancer, cystic cancer, dendritic cancer, cancer à deux, tubular cancer, dyeworkers' cancer, encephaloid tumor, armor-shaped cancer, endometrial cancer, endothelial cancer, epithelial cancer, adenocarcinoma, carcinoma in situ, kang cancer, kangri cancer, latent cancer, medullary cancer, melanoma, mule-spinners' cancer, non-small cell lung cancer, occult cancer, paraffin cancer, pitch workers' cancer, workers') cancer, scar cancer, schistosomal bladder cancer, scirrhous cancer, lymph node cancer, small cell lung cancer, soft cancer, sooty cancer, spindle cell cancer, swamp cancer, tar cancer, and tubular cancers.

Other cancers that may be targeted include carcinoids (gastrointestinal and bronchial), Castleman's disease, chronic myeloproliferative disorders, clear cell sarcoma of the tendon sheath, Ewing's family of tumors, head and neck cancer, lip and oral cancer, Waldenstrom's macroglobulinemia ( s macroglobulinemia, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasms, Wilms tumor, mycosis fungoides, pheochromocytoma, Sezary syndrome, supratentorial primitive neuroectodermal tumor, tumor of unknown primary site, peritoneal effusion, malignant pleural effusion, trophoblastic neoplasm, and hemangiopericytoma.

In embodiments, the cancer may be selected from the group consisting of basal cell carcinoma, breast cancer, leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell carcinoma, melanoma, and combinations thereof. In embodiments, the cancer comprises melanoma. In embodiments, the cancer comprises non-small cell lung cancer.

Further described herein are methods of stimulating an immune response in a mammalian subject, the method comprising administering to the subject a composition as described herein. In some embodiments, the composition is administered directly to the subject in the same manner as a vaccine. In some embodiments, the compositions described herein can be used to induce an immune response to a tumor antigen, one or more pathogenic organisms, or other antigens described herein.

Drug administration

As used herein, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount of a composition sufficient to treat, ameliorate or prevent a confirmed disease or condition or to exhibit a detectable therapeutic, prophylactic or inhibitory effect. The effect can be detected, for example, by improvement of clinical symptoms, alleviation of symptoms, or by any of the assays or clinical diagnostic tests described herein. The precise effective amount for a subject will depend on the subject's weight, stature, and health; the nature and extent of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutic and prophylactic effective amounts for a given situation can be determined by routine experiments within the skill and judgment of the clinician.

As used herein, the term "treatment" includes preventing a specific disorder or condition, or alleviating one or more symptoms associated with a specific disorder or condition and/or preventing or eliminating one or more symptoms, and/or reducing the severity of the condition and/or slowing down the progression of the condition. A completely successful treatment may eliminate the condition (cure), but cure is not a necessary benchmark for successful treatment. "Prevention" refers to a preventive treatment, and its effectiveness can be shown by comparing the results between a population receiving prevention and a population receiving only a negative control. Effective prevention results in a reduced incidence and/or delayed onset and/or reduced severity compared to a control population. In the case of infection, treatment refers to slowing the spread of infection and/or preventing the spread of infection, and/or reducing or eliminating the amount of infectious agents in the host. In the case of cancer, effective treatment can slow the growth of cancer and/or reduce its harmful effects on patients and/or prolong the life of cancer patients and/or prolong the quality of life of patients; or cause tumor shrinkage or a reduction in the number of cancer cells, or cause cancer elimination. In the case of vaccination, treatment enables the body to mount an immune response that inhibits or prevents future infection by the infectious agent, or reduces the severity or duration of an infection or the negative effects of an infection, if one occurs.

The amount of therapeutic agent may be administered as a dose measured in micrograms or milligrams. For purposes of this paragraph,

"Therapeutic" refers to the amount of Compound A in the dose. Contemplated doses of the disclosed therapeutic agents include doses in the range of about 0.1 μg to about 50,000 μg (50 mg).

In the broad guidelines above, dose subranges are specifically contemplated. For example, a dose range for Compound A is contemplated, wherein the lower limit dose of Compound A is any of the following: about 0.5 μg, or about 1 μg, or about 0.5 μg, or about 1 μg, or about 5 μg, or about 10 μg, or about 20 μg, or about 30 μg, or about 40 μg, or about 50 μg, or about 60 μg, or about 70 μg, or about 80 μg, or about 90 μg, or about 100 μg, or about 110 μg, or about 120 μg, or about 130 μg, or about 140 μg, or about 150 μg, or about 160 μg, or about 170 μg, or about 180 μg, or about 190 μg, or about 200 μg, or about 210 μg, or about 220 μg, or about 230 μg, or about 240 μg, or about 250 μg, or about 260 μg, or about 270 μg, or about 280 μg, or about 290 μg, or about 300 μg, or about 310 μg, or about 320 μg, or about 330 μg, or about 340 μg, or about 350 μg, or about 360 μg, or about 370 μg, or about 380 μg, or about 390 μg, or about 400 μg, or about 410 μg, or about 420 μg, or about 430 μg, or about 440 μg, or about 450 μg, or about 460 μg, or about 470 μg, or about 480 μg, or about 490 μg, or about 500 μg, or about 510 μg, or about 520 μg, or about 530 μg, or about 540 μg, or about 550 μg, or about 560 μg, or about 570 μg, or about 580 μg, or about 590 μg, or about 600 μg, or about 650 μg, or about 700 μg, or about 750 μg, or about 800 μg, or about 900 μg, or about 950 μg, or about 1 mg, or about 2 mg, or about 3 mg, or about 4 mg, or about 5 mg, or about 6 mg, or about 7 mg, or about 8 mg, or about 9 mg, or about 10 mg, or about 11 mg, or about 12 mg, or about 13 mg, or about 14 mg, or about 15 mg, or about 16 mg, or about 17 mg, or about 18 mg, or about 19 mg, or about 20 mg, or about 21 mg, or about 22 mg, or about 23 mg, or about 24 mg, or about 25 mg, or about 26 mg, or about 27 mg, or about 28 mg, or about 29 mg, or about 30 mg; and the upper limit dose of Compound A (higher than the selected lower limit dose) is any of the following: about 0.5 μg, or about 1 μg or about 0.5 μg, or about 1 μg, or about 5 μg, or about 1 0 μg, or about 20 μg, or about 30 μg, or about 40 μg, or about 50 μg, or about 60 μg, or about 70 μg, or about 80 μg, or about 90 μg, or about 100 μg, or about 110 μg, or about 120 μg, or about 130 μg, or about 140 μg, or about 150 μg, or about 160 μg, or about 170 μg, or about 180 μg, or about 190 μg, or about 200 μg, or about 210 μg, or about 220 μg, or about 230 μg, or about 240 μg, or about 250 μg, or about 260 μg, or about 270 μg, or about 280 μg, or about or about 470 μg, or about 480 μg, or about 490 μg, or about 500 μg, or about 510 μg, or about 520 μg, or about 530 μg, or about 540 μg, or about 550 μg, or about 560 μg, or about 570 μg, or about 580 μg, or about 590 μg, or about 600 μg, or about 610 μg, or about 620 μg, or about 630 μg, or about 640 μg, or about 650 μg, or about 660 μg, or about 670 μg, or about 680 μg, or about 690 μg, or about 700 μg, or about 710 μg, or about 720 μg, or about 730 μg, or about 740 μg, or about 750 μg, or about 760 μg, or about 770 μg, or about 780 μg, or about 790 μg, or about 560 μg, or about 570 μg, or about 580 μg, or about 590 μg, or about 600 μg, or about 650 μg, or about 700 μg, or about 750 μg, or about 800 μg, or about 900 μg, or about 950 μg, or about 1 mg, or about 2 mg, or about 3 mg, or about 4 mg, or about 5 mg, or about 6 mg, or about 7 mg, or about 8 mg, or about 9 mg, or about 10 mg, or about 11 mg, or about 12 mg, or about 13 mg, or about 14 mg, or about 15 mg, or about 16 mg, or about 17 mg, or about 18 mg, or about 19 mg, or about 47mg, or about 48mg, or about 49mg, or about 50mg.

Doses and dose ranges at the higher end of this range, e.g., a minimum dose of about 1 mg, or about 5 mg, or about 10 mg, or about 15 mg, or about 20 mg, or about 25 mg, or about 30 mg, can be used for oncology indications; doses and dose ranges at the lower end of the above spectrum, e.g., a maximum dose of about 10 mg, can be used for non-oncology indications.

Specific dosage ranges in μg include about 1 μg to about 20000 μg, about 2 μg to about 10000 μg, about 5 μg to about 2000 μg, about 5 μg to about 1000 μg, about 10 μg to about 1000 μg, about 20 μg to about 500 μg, about 30 μg to about 200 μg, and about 50 μg to about 100 μg. The dosage can be the total amount given to the subject per day, or any single dose. Therefore, the dosage can be administered in a single dose or divided doses in a day (e.g., administered in two, three, four or five doses over the course of a day).

The dosage of the liposomes administered will vary with the exact composition of the liposomes. In some embodiments, it is contemplated that the liposomes are administered to produce a daily dose of Compound A of about 0.5 μg to about 50 mg, or about 1 μg to about 45 mg, or about 10 μg to about 40 mg, or about 50 μg to about 3 mg, or about 1 μg to about 20 mg, for example, about 0.5 μg, about 1 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about 410 μg, about 420 μg, about 430 μg, about 440 μg, about 450 μg, about 460 μg, about 470 μg, about 480 μg, about μg, about 240μg, about 250μg, about 260μg, about 270μg, about 280μg, about 290μg, about 300μg, about 310μg, about 320μg, about 330μg, about 340μg, about 350μg, about 360μg, about 370μg, about 380μg, about 390μg, about 400μg, about 410μg , about 420μg, about 430μg, about 440μg, about 450μg, about 460μg, about 470μg, about 480μg, about 490μg, about 500μg, about 510μg, about 520μg, about 530μg, about 540μg, about 550μg, about 560μg, about 570μg, about 580μg, about 590μg, about 600μg, about 610μg, about 620μg, about 630μg, about 640μg, about 650μg, about 660μg, about 670μg, about 680μg, about 690μg, about 700μg, about 710μg, about 720μg, about 730μg, about 740μg, about 750μg, about 760μg, about 770μg, about 7 80μg, about 790μg, about 800μg, about 810μg, about 480μg, about 830μg, about 840μg, about 850μg, about 860μg, about 870μg, about 880μg, about 890μg, about 900μg, about 910μg, about 920μg, about 930μg, about 940μg, about 950μg, about 960 , about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, or about 40 mg. In some embodiments, the liposomes are administered to produce a daily dose of non-glycosidic ceramide of about 1 μg to about 200 μg, or about 1 mg to about 10 mg, or about 700 μg to about 2 mg, or about 800 μg to about 28 mg, or about 10 μg to about 40 mg, about 10 μg to about 5 mg. In some embodiments, the liposomes are administered in divided doses 1 to 4 times per day or in a sustained release form.

The modifier "about" when applied to a numerical value should be understood to include a level of variation at least commensurate with traditional means of measurement of the parameter in question, or 10%, whichever is greater.

The dosage of the therapeutic agent can be alternately administered in a dosage measured in mg/kg (mg compound per kg body weight of the subject being treated). For the purposes of this paragraph, "therapeutic" refers to the amount of Compound A in the dosage. The envisioned mg/kg dosage of the disclosed therapeutic agent includes about 0.00001 mg/kg to about 1 mg/kg. The specific range of dosage in mg/kg includes about 0.0001 mg/kg to about 0.5 mg/kg, about 0.0005 mg/kg to about 0.2 mg/kg, about 0.001 mg/kg to about 0.1 mg/kg, C. All dosages within these ranges are contemplated, including the following: 0.00001 mg/kg, 0.00005 mg/kg, 0.0001 mg/kg, 0.0005 mg/kg, 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg and 1 mg/kg.

The dosage of the therapeutic agent may alternatively be administered in a dosage measured in mg/ ^m2 (mg compound per ^m2 of surface area of the subject being treated). To provide a frame of reference, the average or typical body surface area of an adult female is 1 ^m2 and the average or typical body surface area of an adult male is 1.9 ^m2 , and these numbers can be used to convert/express any of the foregoing dosages or dosage ranges into dosages expressed in mg/ ^m2 . Exemplary contemplated mg/ ^m2 dosages of the disclosed therapeutic agents include about 0.5 mg/ ^m2 to about 20 mg/ ^m2 ; about 1 mg/ ^m2 to about 15 mg/ ^m2 ; about 2 mg/ ^m2 to about 15 mg/ ^m2 ; about 3 mg/ ^m2 to about 10 mg/kg; or about 4 mg/ ^m2 to about 10 mg/ ^m2 .

Combination therapy

The methods disclosed herein may also include treating disease conditions using a composition as described herein together with one or more additional therapeutic agents. Thus, for example, the combination of active ingredients may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by any other combined treatment regimen known in the art. When delivered in alternation therapy, the methods described herein may include sequential administration or delivery of active ingredients, such as in separate solutions, emulsions, suspensions, tablets, pills or capsules, or by different injections in separate syringes. Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e. sequentially, while in combined therapy, an effective dose of two or more active ingredients is administered together, which may be co-formulated or administered separately at substantially the same time. Various sequences of intermittent combined therapy may also be used. In some cases, the compositions disclosed herein are administered and/or formulated together with a second therapeutic agent. In some embodiments, the compositions disclosed herein and further treatment or therapy are administered simultaneously. In some embodiments, the compositions disclosed herein and further treatment or therapy are administered separately.

The second therapeutic agent can be one or more of a chemotherapeutic agent or an immunotherapeutic agent. In some specific cases, the second therapeutic agent is a cytokine, an anti-inflammatory agent, a cancer vaccine, a cancer antigen, or a polynucleotide encoding a cancer antigen. In some cases, the second therapeutic agent is radiation.

Contemplated chemotherapeutic agents for use in the combination therapies disclosed herein include aspirin, sulindac, curcumin, alkylating agents including: nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethyleneimines/methylmelamines such as triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, hexamethylmelamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, arabinose); cytidine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids (including vinblastine (VLB), vincristine and vinorelbine), docetaxel, estramustine, estramustine phosphate; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (erythromycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (miramycin), mitomycin C and actinomycin; and enzymes such as L-asparaginase.

Contemplated biological response modifiers for combination therapy disclosed herein include, but are not limited to, interferon α, IL-2, G-CSF and GM-CSF; various agents, including platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives (including N-methylhydrazine (MIH) and methylbenzalazine), adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; hormones and antagonists, including adrenocortical steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens testosterone, including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; nonsteroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors, and receptor tyrosine kinase inhibitors, such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor), now marketed as Tarveca; and antivirals such as oseltamivir phosphate, amphotericin B, and palivizumab.

Immunotherapeutic agents contemplated for use in the combination therapies disclosed herein, such as immune checkpoint inhibitors. Immunotherapeutic agents include, but are not limited to, Her2/neu receptor antibodies (such as trastuzumab (as commercially available)), anti-CD52 antibodies (such as alemtuzumab (as or Campath-1H), anti-CD33 antibodies (such as gemtuzumab linked to calicheamicin (as commercially available)), anti-CD20 antibodies (such as rituximab (as and Commercially available), Ibritumomab (as commercially available)), anti-TNFα antibodies (such as infliximab (as Commercially available) or adalimumab (as commercially available)), soluble TNFR2 molecules (such as etanercept (as commercially available)), antibodies against the CD25 chain of the IL-2 receptor (such as basiliximab (as commercially available)), anti-CD40/CD40L antibodies (such as humanized IgG1 anti-human CD40 antibody (SGN-40), anti-CTLA-4 blocking antibodies (such as iplimumab (marketed as MDX-101 or MDX-010) or tesimumab), anti-programmed death protein 1 (PD-1) antibodies (i.e., anti-CD279 antibodies), anti-programmed cell death ligand (PDL-1) antibodies, anti-glucocorticoid-induced TNFR-related gene (GITR) antibodies, anti-OX-40 (CD134) antibodies, soluble lymphocyte activation gene 3 (also known as LAG3 or CD223)-based immunomodulators (such as LAG3-Ig (IMP321)), Toll-like receptor agonists (such as monophosphoryl lipid A ), CpG, single-stranded RNA, nucleotides, nucleotide analogs, CL087 (TLR7-specific ligand), loxoribine, polyinosinic-polycytidylic acid, flagellin, resiquimod, imiquimod, gademod, NOD ligands (such as muramyl dipeptide, muramidate, peptidoglycan and muramyl dipeptide).

In an embodiment, the method or use disclosed herein further comprises administering or using at least one checkpoint inhibitor to a subject. In an embodiment, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor may comprise at least one PD-1 inhibitor. In an embodiment, at least one PD-1 inhibitor may be selected from pembrolizumab, nivolumab, cemiplizumab, and spartalizumab. In an embodiment, at least one PD-L1 inhibitor may be selected from atezolizumab, avelumab, and durvalumab. In an embodiment, at least one CTLA-4 inhibitor may be selected from ipilimumab and tesimumab.

In some embodiments, the immune checkpoint inhibitor is an agent that targets the programmed cell death protein 1 (PD-1) pathway. Programmed cell death protein 1 (PD-1), also known as cluster of differentiation 279 (CD279), is a cell surface co-inhibitory receptor expressed on activated T cells, B cells, and macrophages, and is a component of immune checkpoint blockade (Shinohara et al., Genomics., 23 (3): 704, (1994); Francisco et al., Immunol Rev., 236: 219, (2010)). PD-1 limits the activity of T cells when interacting with its two ligands, PD-L1 (also known as B7-H1; CD274) and PD-L2 (B7-DC; CD273) (Postow et al., J Clin Oncol., 33:9, (2015). The interaction of PD-1 with PD-L1 and PD-L2 reduces T cell proliferation, cytokine production, and cytotoxic activity (Freeman GJ et al., J Exp Med., 192:1027–34, (2000); Brown JA et al., J Immunol., 170:1257–66, (2003)).

Agents targeting the PD-1/PD-L1 pathway

Various agents that inhibit the PD-1/PD-L1 pathway described in the art and can be used in the methods disclosed herein. PD-1/PD-L1 inhibitors include, for example, antibodies against PD-1, PD-L1 and PD-L2, PD-1/PD-L1 peptides, PD-1/PD-L1 small molecule compounds, such as (Jiang et al., Front Immunol. 2020, 12; 11: 339; Guo et al. Front. Immunol., 2020; 11: 1508).

PD-1 Antibodies

and 10,752,687 (all of which are incorporated by reference in their entireties).

It is contemplated that any known antibody may be used in the methods of the present invention. In some embodiments, a murine monoclonal anti-target antibody to human PD-1 is used in the methods of the present invention. For example, in vivo MAb anti-human PD-1 (BioXCell, clone: RMP1-14, catalog number: BE0146). Anti-PD-1 antibodies have been shown to be effective in human treatment, see, for example, pembrolizumab ( Merck Sharp & Dohme Corp.), nivolumab ( Bristol-Myers Squibb) and cemiplimab ( Regeneron Pharmaceuticals), which are anti-PD-1 antibodies approved for human treatment. These antibodies all bind to the PD-1 receptor and block its interaction with its ligands PD-L1 and PD-L2. Exemplary FDA-approved anti-PD-1/L1 antibodies are listed in Table PD-A, and anti-PD-1/L1 antibodies under development and patents describing them are listed in Table PD-B.

Additional PD-1 antibodies are being considered for clinical development. For example, pilizumab (CT-011) (CureTech Ltd.); SSI-361 (Lyvgen) (Wang et al., U.S. Patent Application Publication No. 20180346569A1); Prolgolimab (BCD-100) (Tjulandin et al., Ann Oncol. 2019; 30(suppl_11):xi33-xi47); Spartalizumab (PDR001) (Norvatis) (Naing et al., J Immunother Cancer. 2020; 8(1):e000530); GLS-010 (Song et al., Blood (2020) 13(Supplement 1):17); Carrelizumab (SHR1210) (Jiangsu HengRui Medicine Co., Ltd.) (Markham et al., al., 2019 Drugs. 79(12):1355–1361); sintilimab (IBI308) (Innovent and Eli Lilly) (Xu et al., Journal of Clinical Oncology 36(15_suppl):e15125-e15125); tislelizumab (BGB-A317) ("Library Association-Annual Meeting". The Library. s1-1(1):215.1889-01-01); toripalimab (JS 001) (Wang et al., Journal of Clinical Oncology 2019 37:15_suppl,6017-6017); dotalimab (TSR-042, WBP-285) (GlaxoSmithKline) (clinical trial identifier: NCT03981796); INCMGA00012 (MGA012) (Press release: Incyte and MacroGenics, MacroGenics Announces Development Milestone Achieved in retifanlimab (MGA012) Collaboration with Incyte, Source: MacroGenics Inc.); AMP-224 (AstraZeneca/MedImmune and GlaxoSmithKline) (Floudas et al., Clin Colorectal Cancer. 2019); and MEDI-0680 (AMP-514) (AstraZeneca) (Naing et al., J Immunother Cancer. 2019;7(1):225).

PD-L1 Antibodies

Antibodies to PD-L1 have been described, for example, in the following U.S. Patent Nos.: 8217149, 8,779,108, 9,637,546, 10,435,470, 10,208,119, 10,689,445, 10,604,581, 10,775,383, 10,759,856, 10,889,648 (all of which are incorporated by reference).

For example, anti-PD-L1 antibodies have been shown to be effective in human therapy. See, e.g., atezolizumab ( Genentech/Roche); Avelumab ( Merck and Pfizer); and durvalumab ( AstraZeneca), which is an anti-PD-L1 antibody approved for use in humans to treat certain cancers.

Additional PD-L1 antibodies are also specifically considered for use in clinical development. For example, KN035 (Zhang et al., Cell Discovery. 3(1):17004); CK-301 (Checkpoint Therapeutics) (Press Release: Checkpoint Therapeutics Initiates Registrational Development Programs for Anti-PD-L1 Antibody CK-301. Source: Checkpoint Therapeutics, Inc.); BMS-936559 (MDX1105) (Bristol-Myers Squibb/Medarex Inc); Pamilimab (CX-072) (CytomX Therapeutics) (Guo et al. Front. Immunol., 2020; 11:1508); Sugelimab (WBP-3155, CS1001) (CStone Pharmaceuticals) (Guo et al. Front. Immunol., 2020; 11:1508); al. Front. Immunol., 2020;11:1508); and coxilimab (CK-301) (ClinicalTrials.gov identifier: NCT03212404).

PD-L2 Antibodies

Antibodies to PD-L2 have been described, for example, in the following U.S. Patent Nos.: 9,845,356, 10,370,448, and 10,647,771 (incorporated by reference in their entireties).

Table PD-A: FDA-approved anti-PD-1/L1 antibodies (adapted from Guo et al. Front. Immunol., 2020; 11: 1508, incorporated by reference in its entirety).

Each of the cited patents is incorporated herein by reference in its entirety, including its description of the above-described antibodies and their uses.

Table PD-B: These patents are related to anti-PD-1/L1 antibodies currently under development (reprinted from Guo et al. Front. Immunol., 2020; 11: 1508).

Each of the cited patents is incorporated herein by reference in its entirety, including its description of the above-described antibodies and their uses.

PD-1/PD-L1 peptide and small molecule inhibitors

Various PD-1/P-L1 peptides and small inhibitors are contemplated as examples. For example, AUNP12 (PD-1/PD-L1 inhibitor) (Aurigene and Laboratoires Pierre Fabre) (Juneja et al., 2017 The Journal of Experimental Medicine. 214(4):895–904); JTX-4014 (Jounce Therapeutics) (clinical trial identifier: NCT04549025); CA-170 (PD-L1 and VISTA antagonist) (Okazaki et al., (2006). Trends in Immunology. 27(4):195-201); BMS-986189 (macrocyclic peptide) (Bristol-Myers Squibb Pharmaceutical Research Institute) ("Pharmacokinetics, Safety, Tolerability and Pharmacodynamics of BMS-986189 in Healthy Subjects", retrieved from clinicaltrials.gov); JQ1 (BET brominated dopamine inhibitor) (Han et al., Am J Cancer Res 2020; 10(3):727-742); PD-1/PD-L1 macrocyclic peptide (BMSpep-57) (Ganesan et al., Scientific Reports 9, Article number: 12392 (2019); (BMS-103, BMS-142) (Ganesan, et al., Scientific Reports 9, Article number: 12392 (20 19); BMS116 (Chen et al., 2020, Oncoimmunology, 9:1, 1831153).

Table PD-C (10): Small molecule inhibitors of PD-1 and PD-L1 (reprinted from Guo et al. Front. Immunol., 2020; 11: 1508) and related patent documents, incorporated herein by reference.

Table 10 Patents and patent applications for PD-1 and PD-L1 small molecule inhibitors

In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibody against the T cell receptor protein cytotoxic T lymphocyte-associated protein 4 (CTLA-4). An example of an anti-CTLA-4 antibody is the human IgG2 monoclonal antibody tesimumab, which binds to CTLA4 and blocks the binding of antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, thereby inhibiting the downregulation of B7-CTLA4-mediated T cell activation. Another example of an anti-CTLA-4 antibody is the human IgG1 monoclonal antibody ipilimumab, which binds to CTLA4 and blocks the binding of antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, thereby inhibiting the downregulation of B7-CTLA4-mediated T cell activation. Ipilimumab is undergoing clinical trials for non-small cell lung cancer, small cell lung cancer, and metastatic hormone-refractory prostate cancer.

In some embodiments, the anti-GITR antibody is a monoclonal antibody against glucocorticoid-induced tumor necrosis factor receptor (GITR) that blocks the interaction of GITR with its ligand, enhances the cytotoxicity of natural human killer cells and/or downregulates GITR expression on peripheral blood lymphocytes.

In some embodiments, the anti-OX40 antibody is an agonistic monoclonal antibody that mimics the natural OX40 ligand and selectively binds to and activates the OX40 receptor. Receptor activation induces proliferation of memory and effector T cells.

Cytokines

Cytokines that can effectively inhibit tumor growth/metastasis are also considered for use in combination therapy. Such cytokines, lymphokines or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor and erythropoietin.

Cancer vaccines and tumor antigens

The immunotherapeutic agent can be a cancer vaccine. A cancer vaccine is an agent, molecule, or immunogen that stimulates or elicits an endogenous immune response in an individual or subject to one or more tumor antigens.

As used herein, cancer antigens are broadly defined as antigens specifically expressed by tumor or cancer cells: a cancer antigen that is present on the surface of an individual's cancer cells but not on the surface of an individual's normal somatic cells, i.e., the antigen is exposed to the immune system of cancer cells but not in normal somatic cells. Antigens can be expressed on the cell surface of tumor cells, where they are recognized by components of the humoral immune system, such as B lymphocytes (B cells). Intracellular tumor antigens are processed into shorter peptide fragments that form complexes with major histocompatibility complex (MHC) molecules and are presented on the cell surface of cancer cells, where they are recognized by T cell receptors (TCRs) of T lymphocytes (T cells). Preferably, cancer antigens are antigens that are not expressed by normal cells, or at least not expressed to the same level as in tumor cells. Immunotherapeutic agents, such as cancer vaccines, may consist of one or more epitopes or antigenic determinants, such as peptide epitopes or antigenic determinants from tumor or cancer antigens, so that the immune response generated by the cancer vaccine reacts with the antigen.

Cancer vaccines can enhance the presentation of one or more cancer antigens to antigen presenting cells (e.g., macrophages and dendritic cells) and/or other immune cells (such as T cells, B cells, and NK cells). In some instances, the formulations and/or preparations of cancer vaccines can be used together with one or more adjuvants well known in the art to induce an immune response or increase an immune response.

For example, cancer antigens may include testicular cancer antigens encoded by cancer-germline genes. Testicular cancer (CT) antigens constitute a unique group of genes, which are mainly expressed in human germline cells such as placenta and testicles, but are reactivated in various malignant tumors (Simpson et al., Nature Rev (2005) 5, 615-625). Most of these genes are located in a multigene family on chromosome X, and are also referred to as CT-X antigens (Simpson et al., Nature Rev (2005) 5, 615-625). Their expression patterns during embryonic maturation and tumorigenic transformation are similar, thus indicating that they are involved in several steps of tumorigenesis (Simpson et al., Nature Rev (2005) 5, 615-625). CT-X antigens are widely expressed in a variety of cancer types (including, for example, bladder cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, brain cancer, glioma, glioblastoma, hepatocellular carcinoma and melanoma). In addition, their expression patterns are closely associated with advanced disease and poor outcome and may therefore have diagnostic and/or prognostic relevance (Gure et al., Clin Cancer Res (2005) 11, 8055-8062; Velazquez et al., Cancer Immun. (2007) 7(1):11; Andrade et al., Cancer Immun (2008) 8(1):2; Tinguely et al., Cancer Science (2008) 99(4):720-25; Napoletano et al., Am. J. of Obstet. Gyn. (2008) 198:99e91-97). Due to their highly restricted expression in malignant tissues, their tumor-associated peptide epitopes provide promising targets for anticancer immunotherapy (Scanlan et al., Immunol Rev (2002) 188, 22-32). In fact, clinical trials evaluating the role of CT antigens (i.e., MAGE-A3, Prame, and NY-ESO-I) as specific immunotherapy targets have been initiated in many different malignancies (Bender et al., Cancer Immunol (2007) 7, 16; Atanackovic et al., PNAS (2008) 105, 1650-1655; Jager et al., PNAS (2006) 103, 14453-14458; van Baren et al., J Clin Oncol (2005) 23, 9008-9021; Valmori et al., PNAS (2007) 104, 8947-8952; Odunsi et al., PNAS (2007) 104, 12837-12842; Davis et al., PNAS (2006) 103, 14453-14458). al., PNAS (2004) 101, 10697-10702 (9-15)). Tumor antigens, which may include the full-length polypeptide sequence or immunogenic fragment of the tumor antigen, or an epitope derived from the full-length polypeptide sequence of the tumor antigen. Tumor antigens include corresponding nucleotide sequences encoding the full-length polypeptide, immunogenic fragment or epitope derived from the full-length polypeptide sequence of the tumor antigen.

A fragment of a cancer antigen is a continuous amino acid residue in the antigen sequence that is shorter than the full-length antigen (i.e., it is composed of fewer amino acid residues). For example, a fragment may contain less than 500, less than 400, less than 300, less than 200, less than 100 amino acids, or less than 50 amino acids. A fragment will generally consist of at least 5 amino acids, for example, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, or at least 35 amino acids. A fragment of a cancer antigen may contain an immunogenic region or epitope that is bound to an MHC class I or class II molecule and recognized by the TCR of a T lymphocyte. Many such cancer antigen epitopes are known in the art, such as listed on the webpage www.canceramimmunity.org/peptidedatabase/Tcellepitopes.

Cancer antigens can be tumor-associated peptides or proteins that induce or enhance an immune response and are derived from tumor-associated genes and encoded proteins, including, for example, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33, and MAGE-2. /MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brain glycogen phosphorylase, melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, α-lectin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek- can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2 and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, /Lage-2, SP17 and TRP2-Int2, (MART-I), gp100(Pmel17), TR P-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO(LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TAG72, TLP and TPS. For example, the antigenic peptides with tumor characteristics include those listed in International Patent Application Publication No. WO 2000/020581 and U.S. Patent Application Publication No. 2010/0284965, each of which is incorporated herein by reference. In some embodiments, the antigen is a tumor antigen selected from the group consisting of: MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, and mixtures thereof. In some variants, the cancer antigen is a mammalian protein. In some variants, the cancer antigen is a human protein. In some variants, the full-length protein is used as an antigen. In some variants, peptides comprising antigenic fragments of these proteins are used as tumor antigens.

Other suitable antigens include cancer antigens from the following classes: cancer-testis antigens (e.g., HOM-MEL-40), differentiation antigens (e.g., HOM-MEL-55), overexpressed gene products (HOM-MD-21), mutant gene products (NY-COL-2), splice variants (HOM-MD-397), gene amplification products (HOM-NSCLC-11), and cancer-associated self-antigens (HOM-MEL-2.4), see Cancer Vaccines and Immunotherapy (2000) Stern, Beverley and Carroll, eds., Cambridge University Press, Cambridge. Further examples include MART-1 (melanoma antigen recognized by T cells-1), MAGE-A (MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A12), MAGE B (MAGE-B1-MAGE-B24), MAGE-C (MAGE-C1/CT7, CT10), GAGE (GAGE-1, GAGE-8, PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE (LAGE-1a(1S), -1b(1L), NY-ESO-1), SSX (SSX1-SSX-5), BAGE, SCP-1, PRAME (MAPE), SART-1, SART-3, CTp11, TSP50, CT9/BRDT, gp100, MART-1, TRP-1, TRP-2, melan-A/MART-1, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), MUCIN (MUC-1), and tyrosinase. TAA is reviewed in Cancer Immunology (2001) by Kluwer Academic Publishers, The Netherlands. Other cancer-associated antigens include Her 2, survivin, and TERT. In some embodiments, the tumor antigen comprises NY-ESO-1 or an antigenic fragment thereof.

The term "antigen" refers to a protein or peptide to be introduced into a subject. As described herein, an antigen can be provided by delivering a peptide or protein or by delivering a nucleic acid encoding a peptide or protein.

In the context of the present disclosure, "antigen" also means an antigenic peptide derived from an antigen. In particular, "cancer associated antigen" is intended to include peptides derived from a cancer associated antigen.

An antigen, such as a cancer associated antigen, can be provided in several different ways for use as a medicament. It can be administered as part of a carrier.

Any suitable vector can be used to introduce a polynucleotide encoding a polypeptide of the present invention, which encodes one of the tumor antigen proteins, into a host. Other vectors described in the literature include replication-defective retroviral vectors, including but not limited to lentiviral vectors [Kim et al., J. Virol., 72(1):811-81 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp.43-46.]; adeno-associated virus (AAV) vectors [U.S. Pat. No. 5,474,935, U.S. Pat. No. 5,139,941, U.S. Pat. No. 5,622,856, U.S. Pat. No. 5,658,776, U.S. Pat. No. 5,773,289, U.S. Pat. No. 5,789,390, U.S. Pat. No. 5,834,441, U.S. Pat. No. 5,863,541, U.S. Pat. No. 5,851,521, U.S. Pat. No. 5,252,479, Gnatenko et al., J. Invest. Med., 45:87 98 (1997)]; adenovirus (AV) vectors [see, e.g., U.S. Pat. No. 5,792,453, U.S. Pat. No. 5,824,544, U.S. Pat. No. 5,707,618, U.S. Pat. No. 5,693,509, U.S. Pat. No. 5,670,488, U.S. Pat. No. 5,585,362, Quantin et al., Proc. Natl. Acad. Sci. USA, 89:2581 2584 (1992); Stratford Perricadet et al., J. Clin. Invest., 90:62630 (1992); and Rosenfeld et al., Cell, 68:143 155 (1992)]; adenovirus-adeno-associated virus chimeras (see, e.g., U.S. Pat. No. 5,856,152) or vaccinia virus or herpes virus (see, e.g., U.S. Pat. No. 5,879,934, U.S. Pat. No. 5,849,571, U.S. Pat. No. 5,830,727, U.S. Pat. No. 5,661,033, U.S. Pat. No. 5,328,688, liposome-mediated gene transfer (BRL); liposome vectors [see, e.g., U.S. Pat. No. 5,631,237 (liposomes containing Sendai virus proteins)]; and combinations thereof.

Suitable cancer vaccines are known in the art and may be produced by any convenient technique.

For example, cancer vaccines can be generated in whole or in part by chemical synthesis. For example, peptide-based vaccines or immunogens can be synthesized using liquid or solid phase synthesis methods; synthesized in solution; or synthesized by any combination of solid phase, liquid phase and solution chemistry, for example, by first completing the corresponding peptide portion and then, if necessary and appropriate, after removing any protecting groups present, introducing residue X by reaction of the corresponding carbonic or sulfonic acid or its reactive derivatives. The chemical synthesis of peptides is well known in the art (J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984); M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J.H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford ford1991; in Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California; G.A. Grant, (Ed.) Synthetic Peptides, A User’s Guide. W.H. Freeman & Co., New York 1992, E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press 1989 and in G. B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289)). Academic Press, New York and London 1997).

Alternatively, peptide-based cancer vaccines can be generated in whole or in part by recombinant techniques. For example, nucleic acids encoding cancer antigens can be expressed in host cells, and the expressed antigens can be separated and/or purified from cell culture. For example, antigens can be expressed in E. coli in a soluble form or inclusion bodies, which can be dissolved and refolded. After expression, the antigens can be separated and/or purified. Cancer antigens can be analyzed by standard techniques, such as mass spectrometry and Western blot analysis.

The use of tumor antigens to generate an immune response is well established in the art (see, e.g., Kakimi K, et al. Int J Cancer. 2011 Feb 3; Kawada J, Int J Cancer. 2011 Mar 16; Gnjatic S, et al. Clin Cancer Res. 2009 Mar 15; 15(6):2130-9; Yuan J, et al. Proc Natl Acad Sci US A. 2008 Dec 23; 105(51):20410-5; Sharma P, et al. J Immunother. 2008 Nov-Dec; 31(9):849-57; Wada H, et al. Int J Cancer. 2008 Nov 15; 123(10):2362-9; Diefenbach CS, et al. Clin Cancer Res. 2008 May 1; 14(9):2740-8; Bender A, et al. Cancer Immun. 2007 Oct 19; 7: 16; Odunsi K, et al. Proc Natl Acad Sci US A. 2007 Jul 31; 104(31): 12837-42; Valmori D, et al. cerImmun.2007Apr 19;7:9;Kawabata R,et al.Int J Cancer.2007May15;120(10):2178-84; E, et al. Proc Natl Acad Sci US A. 200 Sep 26; 103 (39): 14453-8; Davis ID Proc Natl Acad Sci US A. 2005 Jul 5; 102 (27): 9734; Chen Q, Proc Natl Acad Sci U SA. 2004 Jun 22; 101 (25): 9363-8; E, Proc Natl Acad Sci US A. 2000 Oct 24; 97(22): 12198-203; Carrasco J, et al. J Immunol. 2008 Mar 1; 180 (5): 3585-93; van Baren N, et al. Int JCancer.2005Nov20; 117(4):596-604; Marchand M, et al. Eur J Cancer.2003Jan; 39(1):70-7; Marchand M et al. 5;105(5):1650-5).

Typically, an immunotherapeutic agent (e.g., a cancer vaccine) is administered to an individual whose cancer expresses the antigen. Cancer cells from an individual can be analyzed to identify the cancer antigen, and then the patient is identified to administer an appropriate immunotherapeutic agent or cancer vaccine. For example, the methods described herein may include a step of confirming a cancer antigen that is displayed by one or more cancer cells in a sample obtained from an individual.

Biological samples, such as biopsies, blood or bone marrow samples, can be obtained from the subject and tested for the presence of cancer cells, which can be identified using any standard technique (including but not limited to immunotechniques, such as immunocytochemistry and immunohistochemistry) to show cancer antigens. Other techniques include immunological analysis, such as serological determination of autoimmune responses to the cancer antigens, see WO2001/007917. Analysis of gene expression can be performed using methods known in the art, such as polymerase chain reaction or microarray analysis.

Cancer vaccines can be administered in combination with adjuvants, including those described above. Other suitable adjuvants include aluminum salts, such as alum (potassium aluminum sulfate dodecahydrate), aluminum hydroxide, and aluminum phosphate, and organic compounds, such as squalene.

In addition to the cancer antigen, the immunotherapeutic preparation, immunogenic preparation or vaccine preparation may contain an adjuvant. For example, in the above-mentioned pharmaceutically acceptable carrier or diluent, the preparation may contain 1-500 μg, preferably 1-50 μg, of the cancer antigen and 0.5-20 mg, preferably 1-10 mg of the adjuvant.

Vaccine formulations may include Toll-like receptor (TLR) ligands. Suitable TLR ligands include polyinosinic acid (polyI:C), lipopolysaccharide (LPS), CpG oligonucleotides, poly-LC, poly-ICLC, MPL (Corixa Corp) and imidazoquinolines, such as imiquimod and R848. It is well known in the art that TLR ligands are used to regulate immune responses (see, e.g., Weineret al (1997) PNAS USA 94 10833-10837; Vabulas et al J. Immunol. (2000) 164 2372-2378; Gunzer et al (2005) Blood 10 2424-2432).

Formulations of immunotherapeutic agents, such as cancer vaccines, are well known in the art and include MAGE-A3 ASCI, NY-ESO-1 ASCI, and PRAME ASCI (GSK Bio); Provenge (Dendreon), Abogovomab (Meranini), M-Vax (Avax), Alloceptin-7 (Vial) for metastatic melanoma, GSK1572932A (GSKBio) Belagenpumatucel-L (Novarex) BMP-25 (Merck Serono), BiovaxID (Biovest/Accentia), MDX-1379 (Medarex/BMS), Ipilimumab (BMS) Trovax (Oxford Biomedical) bacteriophage (Antigenics), and PR1 leukemia peptide (The Vaccine Company).

Antibody combination therapy

Embodiments involving the use of antibodies as co-therapeutic agents may include all classes of antibodies that have been developed for this purpose, including but not limited to monoclonal antibodies, chimeric antibodies, and antibody fragments.

"Monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies. Monoclonal antibodies are generally highly specific and can be directed against a single antigenic site, whereas polyclonal antibody preparations typically include different antibodies directed against the same or different determinants (epitopes). In addition to the specificity of monoclonal antibodies, they are also advantageous because they are synthesized by homogeneous cultures and are not contaminated by other immunoglobulins with different specificities and characteristics.

Monoclonal antibodies can be made by the hybridoma method originally described by Kohler et al. (Nature, 256:495-7, 1975) (Harlow &Lane; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1988); Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581-597, 1991). Additional methods for producing monoclonal antibodies are well known to those of ordinary skill in the art.

Monoclonal antibodies, such as those produced by the methods described above, are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures including, for example, protein A-Sepharose, hydrophobic interaction chromatography (HIC), ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.

It is also contemplated that the antibodies of the present disclosure may be used as smaller antigen-binding fragments of antibodies known in the art and described herein.

Because chimeric or humanized antibodies are less immunogenic in humans than the parental non-human (eg, mouse) monoclonal antibody, chimeric or humanized antibodies can be used to treat humans with much less risk of allergic reactions.

Chimeric monoclonal antibodies can be produced using standard procedures known in the art (see Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984); and Boulianne et al, Nature 312, 643-646, (1984)) in which the Ig variable domains of a non-human (e.g., mouse) monoclonal antibody are fused to human Ig constant domains.

Humanized antibodies can be achieved by a variety of methods, including, for example: (1) grafting non-human complementarity determining regions (CDRs) onto human framework and constant regions (a process known in the art as humanization via "CDR grafting"); (2) grafting entire non-human variable domains, but "masking" them with a human-like surface by replacing surface residues (a process known in the art as "veneering"); or alternatively, (3) substituting human amino acids at positions determined to be unlikely to adversely affect antigen binding or protein folding, but likely to reduce immunogenicity in a human environment (e.g., HUMAN ENGINEERING ^™ ). In the present disclosure, humanized antibodies will include "humanized" antibodies, "veneering" antibodies, and "HUMAN ENGINEERED ^™ " antibodies. These methods are disclosed in, for example, Jones et al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci., USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-153 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31:169-217 (1994); Studnicka et al. U.S. Patent No. 5,766,886; Studnicka et al., (Protein Engineering 7:805-814, 1994; Co et al., J. Immunol. 152, 2968-297 (1994); Riechmann, et al., Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng. 4:773-783 (1991), each of which is incorporated herein by reference. CDR grafting techniques are known in the art, see, for example, Riechmann, et al. (Nature 332:323-27 (1988).

Human antibodies against target proteins can also be produced using transgenic animals that do not have endogenous immunoglobulins and are engineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals with human Ig loci, wherein the animals do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy chain loci and light chain loci. WO 91/0090 also discloses transgenic non-primate mammalian hosts capable of launching an immune response to an immunogen, wherein the antibody has a primate constant region and/or a variable region, and wherein the loci encoding endogenous immunoglobulins are replaced or inactivated. WO 96/30498 and U.S. Patent No. 6,091,001 disclose the use of the Cre/Lox system to modify the immunoglobulin loci of mammals, such as replacing all or part of the constant region or the variable region to form a modified antibody molecule. WO94/02602 discloses a non-human mammalian host with an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Pat. No. 5,939,598 discloses a method for preparing a transgenic mouse, wherein the mouse lacks endogenous heavy chains and expresses an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. See also U.S. Pat. Nos. 6,114,598, 6,657,103, and 6,833,268; Green LL, Curr Drug Discovery Technol., 11(1), 74-84, 2014; Lee EC et al., Nature Biotechnology, 32: 356–363, 2014; Lee EC and Owen M, Methods Mol Biol., 901: 137-48, 2012.

The present invention will be more fully understood with reference to the following examples, which detail exemplary embodiments of the present invention. However, they should not be construed as limiting the scope of the present invention. All references in this disclosure are expressly incorporated by reference herein.

Examples

IMM60 is a highly lipophilic molecule (necessary for tight binding to CD1d) and is virtually insoluble in aqueous solution. Therefore, a liposomal formulation is selected so that IMM60 can be delivered in a physically stable and soluble form required for IV administration. A relevant physicochemical factor is that IMM60 has a high calculated logarithm (17.6) of the partition coefficient (cLogP) value, indicating its lipophilicity. Structurally, IMM60 contains 2 straight-chain aliphatic tails. Based on cLogP, IMM60 is more lipophilic than DSPC. Therefore, IMM60 with a long lipid chain is embedded in the liposome bilayer and dissolved as a component of the phospholipid bilayer membrane. Liposomal formulations can specifically target tumor sites by enhancing permeability and retention (EPR) effects. Micelles and liposomes containing anticancer drugs are some examples of nanoparticle formulations, in which small colloidal nanoparticles in the range of 100nm to 200nm can be extravasated in the tumor vasculature and penetrate and accumulate in tumor tissues by the EPR effect. In addition, when administered as liposomes, drug concentrations in tumors can be as high as 10- to 30-fold compared to dissolved formulations, and the EPR effect can be seen within 10 minutes after IV injection. Given these physicochemical properties of IMM60 and these practical advantages of liposome-targeted tumors, it is considered to be an ideal vehicle for glycolipids (such as IMM60) for the treatment of cancer.

Example 1 - Preparation of Compositions

Materials and methods

API/Lipid Solvent Solution (Ethanol Solution): API is weighed and dissolved in a water-soluble organic solvent system at temperature, typically requiring 15 minutes of mixing to completely dissolve the API at 60°C. The dissolved API is added to the lipids. To completely dissolve the lipids in the solvent, water may be added to ensure that all lipids are completely dissolved. The placebo batch does not include the API in the solvent solution.

Lipid hydration (MLV formation): The aqueous phase consists of 145 mM sodium chloride and 10 mM sodium phosphate at pH 6.5 or an alternative buffer. After diluting the organic phase containing the API and lipids into the aqueous phase, the lipids are hydrated and multilamellar vesicles (MLVs) are formed. Hydration is allowed to proceed for several minutes at the temperature with mixing.

Extrusion: Set up the Lipex extrusion system and connect the system to a heated water bath to maintain the process temperature and prepare several layers of polycarbonate filters. Place the MLVs into the LIPEX extruder with polycarbonate filters to reduce the size to large unilamellar vesicles (LUVs) with a target vesicle size in the range of 90nm to 100nm in diameter. Nitrogen is used to pressurize the system to 600psi and extrude the MLVs through the polycarbonate membrane filter. Multiple extrusions may be required to achieve the target particle size range.

Diafiltration: After size reduction, LUVs were cooled to near room temperature (<30°C) and loaded onto a diafiltration cartridge. In this process, liposomes were washed using essentially tangential flow and buffer exchanged using 145 mM sodium chloride and 10 mM sodium phosphate at pH 6.5 to remove organic solvents from the formulation. Tangential flow filtration was also used to concentrate the product to adjust lipid and API concentrations to target specifications (to 1 mg/mL API and 9 mg/mL lipid).

Clarification and sterile filtration: After the diafiltration process is complete, the product is filtered through a 0.2 μm sterilizing grade filter to reduce bioburden. Ideally, the product concentration is slightly higher than the value required by the label. After clarification filtration, the product is stored overnight while the lipid and API content is determined in process. The product is diluted to the final concentration required by the label and sterile filtered through 2 × 0.2 μm mPES membrane filters (Sartopore, Sartorius).

Particle size measurement by dynamic light scattering: During the preparation process, the particle size was monitored using dynamic light scattering measurements at two angles, 90° and 12.8°. The product was diluted in saline (0.9% sodium chloride) to a lipid concentration range of 0.5 mg/mL to 1 mg/mL, pre-equilibrated in a disposable sizing cup at 23°C for 2 minutes, and read using a Malvern Zetasizer instrument at the following settings: dispersion refractive index (RI) of 1.332, material RI of 1.50.

Prepare liposomes disclosed herein. Prepare an ethanol solution (e.g., 4.5 mL ethanol and 0.5 mL water) including lipids and API (e.g., 50 mg/mL lipid; 5.2 mg/mL API) and heat to 65°C while stirring. For example, add 4.5 mL ethanol/0.5 mL water to 0.1238 g DSPC, 0.1004 g DSPG, and 0.0272 g IMM60 (API requirement: 0.0259 g API adjusted to 95.3% purity). Add the ethanol solution to a buffer solution (e.g., 250 mM sucrose and 145 mM sodium chloride). For example, add 5 mL of the ethanol solution to 45 mL of a buffer solution heated at 65°C while stirring for at least 5 minutes ([lipid] = 4.5 mg/mL,

[API] = 0.5 mg/mL, solvent = 9%) to form 50 mL of multilamellar vesicle solution. The multilamellar vesicle solution is extruded, for example, at 65°C through a 2×80 nm membrane to form liposomes. The liposomes are immediately diluted with an equal volume of room temperature buffer and ultrafiltered until concentrated to 50 mL ([API] = 0.5 mg/mL). The ultrafiltered solution is diafiltered (e.g., Krosflow) for 10 working volumes (500 mL) using a 50 mL reservoir (e.g., 115 cm ² ). The solution is concentrated to about 1.3 mg/mL API. The solution is filtered at ambient temperature using a clarifying filter (filter 0.2 μm Sartopore (mPES membrane). The filtered solution is then diluted with buffer to the 1 mg/mL API required by the label. The solution is then sterile filtered through a redundant 0.2 μm Sartopore (mPES membrane) sterile filter. The estimated yield is about 85%.

The ethanol solution may contain two lipids selected from the following:

(a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG);

(b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol);

(c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB);

(d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC);

(e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); and

(f) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL).

Development of formulations: According to Table 1, the first liposomal IMM60 prototype was prepared in studies A, B, C and D. Tables 2 and 3 summarize the particle size, API and lipid content after sterile filtration. Initially, the liposomal IMM60 prototype was prepared to evaluate the feasibility of the manufacturing process by incorporating IMM60 into EPC/EPG liposomes (A). The particle size of MLV from batch A was particularly small, approximately 5 nm, and remained stable throughout the process. The API content was below the target value of 1 mg/mL (refer to Table 3). The low API content in the final product is attributed to dilution of the product rather than loss of API during the diafiltration process.

Subsequently, a second batch (B) was prepared in which IMM60 was incorporated into DSPC/DSPG liposomes. Compared to A, the particle size of MLV from batch B was larger, about 178 nm. Although extrusion resulted in size reduction, the particle size of LUV after ultrafiltration/diafiltration was slightly larger (>100 nm). Sterile filtration removed some particles, which was confirmed by the reduction in particle size (about 101 nm). Accordingly, the recovery of API and lipids was lower (see Table 3).

As described in Table 1, seven additional liposomal IMM60 prototypes were prepared by incorporating IMM60 into various formulations (C01 to C07). Of the seven different formulations prepared in C, batches 01-03 and 05-06 were successfully prepared. Prototype C01 resulted in larger particle size (68 nm) and better API incorporation (0.957 mg/mL) compared to the first prototype (A) with the same API and lipid composition (EPC/EPG/IMM60; weight ratio 2.25/6.75/1). Prototypes C02 (POPC, DDAB, IMM60) and C03 (POPC, DC-Chol, IMM60) had API incorporation rates greater than 70% and performed well during the preparation process, although the DF process time for prototype C02 was longer. Batch 05 (DSPC/DSPG) did show some particle size growth and was difficult to sterile filter, resulting in 50% API and lipid losses. Batch 04 and Batch 07 contained DOTAP which appeared to degrade significantly during the dissolution process as the DOTAP content was much lower than expected for MLV (approximately 0.1-0.2 mg/mL). The degradation was likely due to the high temperature (60°C) required in the manufacturing process as the solubility of the API depends on this temperature. Batch 04 contained POPC/DOTAP and showed a slight increase in particle size. Batch 07 contained only cholesterol as the second lipid and showed a significant increase in size. Batch 07 also showed flocculation which resulted in difficulty in filtration and significant loss of lipid and API.

The DOTAP:POPC batch was prepared a second time according to Protocol D, with a shorter temperature exposure time and the use of sucrose buffer instead of saline. This repeated batch showed an improvement in particle size and improved stability of the lipid at each stage of the preparation process.

Table 1: API and lipid composition

batch Composition (wt/wt/wt) A EPC, EPG, IMM60(2.25/6.75/1) B DSPC, DSPG, IMM60(4.78/3.88/1) C01 EPC, EPG, IMM60(2.25/6.75/1) C02 POPC, DDAB, IMM60(4.58/3.04/1) C03 POPC, DC-Chol, IMM60(4.60/2.60/1) C04 POPC, DOTAP, IMM60(4.61/3.40/1) C05 DSPC, DSPG, IMM60(4.76/3.86/1) C06 DMPG, Chol, IMM60(4.60/1.64/1) C07 DOTAP, Chol, IMM60(4.66/1.64/1) D01 POPC, DOTAP, IMM60(4.61/3.40/1)

Table 2: Particle size data

Table 3

From all the prototypes prepared, the lead formulation (C05) was selected based on the efficacy study despite its poor sterile filtration performance. Study E optimized the manufacturing process for this prototype as the final sterile filtration of the product was found to be challenging. The filtration step resulted in filter clogging and subsequent reduction in lipid and API content of the filtered product.

The lead formulation (DSPC, DSPG, IMM60 (weight ratio 5/4/1)) was prepared again at a 5 mL scale (reference batch E) to identify the limitations of the original manufacturing process. During the manufacturing process, single-pass extrusion resulted in an effective size reduction to achieve a vesicle size of approximately 8 nm. However, upon processing through a diafiltration cartridge, the particle size increased. This size increase may result in poor product filtration capabilities.

In-process determination of lipid and API content of Batch E was monitored throughout the manufacturing process. After extrusion, the recovery of lipid and API was 95%, but after diafiltration and ultrafiltration to half of the initial volume, the recovery dropped to 55%. The subsequent filtration step, using a cellulose acetate membrane (0.2 μm sterilizing grade filter), also resulted in significant losses of product, with only 18% of the initial amount recovered. Initial filtration trials with polyethersulfone (PES) instead of cellulose acetate (CA) membranes showed promising results, which were further confirmed during process optimization.

The preparation process was optimized by studies F, G, I, J, and L. The particle size instability issue was first addressed in study F to optimize the LUV particle size after extrusion (cooling rate and solvent concentration). In addition, in order to reduce the particle size increase caused by high solvent concentration during ultrafiltration, diafiltration (DF) was performed before ultrafiltration (UF). However, LUV size increase was still observed throughout the process, so study G further included immediate dilution of the LUV solution by extrusion into cold or hot buffer. The temperature of the buffer did not appear to have a direct effect on the particle size, but diluting the LUV solution before DF did improve the stability of the particle size. Further stabilization of the particle size was achieved by increasing the surface area of the filter cartridge from 20 ^cm2 to 115 ^cm2 , thereby increasing the diafiltration rate.

Study I demonstrated particle size stability when extruded into room temperature buffer (1:1); and lipid recovery was improved by about 9% when using a filter cartridge with a smaller pore size (100 kD vs. 500 kD). A 50 mL batch (Batch J) using IMM60 was prepared using a modified procedure that included post-pressing dilution (1:1) with RT buffer and diafiltration prior to ultrafiltration. Using a filter cartridge with a pore size of 100 KDa for DF/UF was problematic and significantly increased processing time. For this reason, DF/UF was instead performed using a 115 cm ² , 500 kDa filter cartridge.

A production scale of approximately 7 mL was performed in Study L to test a new batch of API using the new manufacturing process. No difficulties were encountered with the API, and particle size, lipids, and API content were close to target.

Aggregation of the liposomes was observed in early stability testing. Adding sucrose to the buffer and including an annealing step during preparation resulted in a smaller increase in particle size and appeared more stable over time compared to the original formulation prepared in a buffer containing saline alone. The results obtained with the DSPC/DSPG/CHOL/IMM60 formulation also suggest that reducing the DSPG content (compensated by adding 20 mol% cholesterol) may slightly improve particle size stability after refrigerated storage at 4° (13 days post-DOM) in a buffer containing sucrose (with or without an annealing step).

The methods and resulting products described in this example and the following examples are considered aspects of the present invention.

Example 2 - Stabilizer Testing

Two compositions disclosed herein were prepared and tested for stability. The first composition (batch 1) was prepared according to Example 1 and formulated according to Table 4. The initial stability test of the first composition included testing the particle size of multilamellar vesicles (MLVs) after liposomes were added to the buffer at a 1:1 dilution (pathway 1), after diafiltration (after DF), after ultrafiltration (after UF), and after clarification filtration (after CF), as shown in Table 5. The average diameter (90°Z average) was initially 102 (T=0), as shown in Table 5. The first composition was then stored in a buffer (250mM sucrose, 145mM sodium chloride, 10mM sodium phosphate, pH 6.5) at 25°C, 5°C, and -20°C. Table 6 shows the concentrations of various components in the composition after 9 days and 1 month. Table 7 shows the average diameter and polydispersity index (PdI) at T=7, 14, 21, 28, 42, 57, 206, 206 during heat treatment and at 231 and 231 days during heat treatment (heating). Heat treatment includes heating the liposomes to 55°C for 10 minutes after storage. Table 7 shows the formulations when stored at -20°C, 5°C and 25°C. Exemplary means for heating include a heating block, a hot plate, an oven and a water bath.

Table 4-DSPC/DSPG/IMM60

Table 5

Table 6

Table 7: Particle size (storage for 7.5 months)

A second composition (Batch 2) was prepared according to Example 1 and formulated according to Table 8. Initial stability testing of the second composition included testing the particle size after MLV, after adding the liposomes to the buffer at a 1:1 dilution (Path 1), after diafiltration (DF), after ultrafiltration (UF), and after clarification filtration (CF), as shown in Table 9. The average diameter (90° Z average) was initially 92.8 (T=0). The second composition was then stored in a buffer (250 mM sucrose, 145 mM sodium chloride, 10 mM sodium phosphate, pH 6.5) at 25°C, 5°C, and -20°C. Table 10 shows the concentrations of the various components in the composition after 8 days and after 1 month. Table 11 shows the average diameter and polydispersity index (PdI) at T=7, 14, 21, 28, 42, 57, 206, and 206 during the heat treatment. The heat treatment included heating the liposomes to 55°C for 10 minutes after storage. Table 11 shows the formulations when stored at -20°C, 5°C and 25°C.

Table 8

Batch 2 [Measurement value] mg/mL Weight Ratio Molar Ratio DSPC 4.20 44.6% 41 DSPG 3.18 33.8% 31 CHOL 0.91 9.7% 18 IMM60 1.12 11.9% 10 Total lipid content 9.41 100.0%

Table 9

Table 10

Table 11: Particle size (storage for 7 months)

These data indicate that the liposome formulations described herein are storable, particularly at relatively low temperatures. These data further indicate that heat treatment of liposomes, particularly stored liposomes, can reduce particle size and PdI. Without intending to be bound by any particular theory, it is contemplated that heat treatment reduces clumping of liposomes.

Compositions comprising heat-treated liposomes are contemplated as an aspect of the present invention. Preparation methods comprising a heat-treatment step are contemplated as an aspect of the present invention. Methods for preparing or standardizing liposome compositions comprising heat treatment are contemplated as an aspect of the present invention, particularly for liposome compositions stored under frozen or sub-zero conditions, or liposome compositions that are freeze-dried and reconstituted in an aqueous buffer. Treatment methods comprising a heat-treatment step are contemplated as an aspect of the present invention. In some embodiments, the time and temperature of the heat treatment are sufficient to reduce the average particle size and/or reduce the PdI of the composition. In exemplary embodiments, the temperature of the heat treatment is in the range of about 55°C to about 65°C. In some embodiments, the heat treatment is for about 5 minutes to about 15 minutes, for example, about 10 minutes.

Biological studies of liposomes containing IMM60

Example 3 - Comparison of liposomal IMM60 and soluble IMM60

Liposomal IMM60 (DSPC:DSPG:IMM60) was compared with soluble IMM60 to understand its ability to generate an ovalbumin-specific immune response and its ability to mature dendritic cells and induce IFN-γ accumulation in serum. Both (soluble or liposomal) IMM60 formulations were co-injected with ovalbumin antigen into C57BL/6 mice. When liposomal IMM60 and soluble IMM60 were injected at a dose of 0.1 ng/mouse (approximately 4 ng/Kg), they induced similar levels of serum IFN-γ accumulation, with no statistically significant difference between the two. However, liposomal IMM60 was more potent against mature splenic dendritic cells 18 hours after administration, and also significantly increased CD8+ T cells specific for the ovalbumin peptide epitope SIINFEKL 7 days after administration.

The early stimulatory capacity of these formulations was tested by measuring serum IFN-γ 18 hours after injection. At this time point, high levels of IFN-γ in serum are a well-established standard assay for iNKT cell activation, and it was used as a surrogate assay for early activation of iNKT cells, which was then cascaded into amplified IFN-γ production by NK cells. The second assay assessed the maturation level of splenic dendritic cells 18 hours after administration of the IMM60 formulations. Previous studies have shown that cognate interactions between iNKT cells and dendritic cells lead to maturation of dendritic cells in a CD40:CD40 ligand-dependent manner. 18 hours after IMM60 injection, 2 animals from each group were taken and the levels of CD86 on the surface of splenic CD11c+ cells, a marker of dendritic cell maturation, were measured. Seven days after injection, the remaining animals within each group were used to assess the number of T cells that specifically recognized an ovalbumin-derived peptide (SIINFEKL). This was done by drawing blood and staining blood lymphocytes with fluorescent tetrameric MHC class I monomers carrying the SIINFEKL peptide.

Mice are the animal model of choice for the in vivo studies described herein because they are the lowest vertebrate group in the evolutionary tree and they have suitable models of immune responses in general and iNKT biology in particular, including mutant strains lacking iNKT cells or CD1d molecules.

Storage and preparation before use:

Liposome IMM60-vials were thawed at room temperature (21°C to 22°C). The vials were then heated to 55°C for 10 minutes using a heated water bath, mixed by inverting 6 times, cooled to room temperature, and diluted to the desired concentration with tissue culture grade PBSA.

Soluble IMM60 - The lyophilized material was resuspended to 10 mg/ml (10 μl contained 100 μg vial) in chloroform: methanol: water (10: 10: 3), spun to capture all the material, and then the vehicle solution (150 mM sodium chloride and 0.5% Tween20 in distilled water) was quickly added to a final concentration of 100 μg/ml (for 10 μl containing 100 μg, 990 μl of vehicle was added). This composition was then heated to 80°C to 90°C for 5 minutes and sonicated for 5 minutes using a water bath sonicator.

Fifty-four C57BL/6J OlaHsd female mice, 7-8 weeks old, were used in this study. The mice were purchased from Envigo and weighed 17-19 grams.

Maxisorb plates 96 flat bottom half size wells (Nunc).

Capture antibody: clone R4-6A2 (eBioscience)

Detection antibody: clone XMG1.2-biotin (eBioscience).

Streptavidin-HRP (Thermo Fisher Scientific)

TMB substrate (Sigma)

Sulfuric acid 0.1N (Sigma)

3. Splenocyte Processing

PBS, RPMI, FBS, Penicillin/Streptomycin (Pen/Strep), Glutanime (all tissue culture grade products from Gibco)

Red blood cell lysis buffer (Qiagen)

All antibodies were purchased from eBioscience or Biolegend CD11b-FITC, CD11b-PEcy7, CD105-BV421, CD45.2-APC, CD11c-APC-cy7, CD86-PE, PD-L1-PE, CD40-PEcy7, CD11c-APC and MHC class II-APCcy7 (from Biolegend). B220-eFluor450 (from eBioscience). Live/dead fixative stain (ThermoFisher scientific)

Anti-CD16/CD32 Fc blocking antibody (eBioscience)

Preparation of reagents:

Ovalbumin 400 μg/100 μl: 20 mg of ovalbumin was reconstituted in 5 ml with PBS (TC grade).

Liposome IMM60 (stock solution 0.47 mg/ml):

First dilution: 4.7 ml PBS + 10 μl stock solution DSPC:DSPG:IMM60

Second dilution: 10 ml PBS + 10 μl first dilution

3rd dilution 9ml PBS + 1ml 2nd dilution

Soluble IMM60 (stock solution 100 μg/ml):

First dilution: 5 ml PBS + 5 μl stock solution

Second dilution: 4.95 ml PBS + 50 μl first dilution

The mice were then placed in a cage in a 37°C heating chamber to pre-warm for 20 to 30 minutes. For Group 1: 200 μl of PBS was injected intravenously. For Group 2: 200 μl of a 1:1 mixture of ovalbumin and PBS. For Group 3: 200 μl of a 1:1 mixture of ovalbumin and soluble IMM60 (2nd dilution). For Group 4: 200 μl of a 1:1 mixture of ovalbumin and liposomal IMM60 (2nd dilution). For Group 5: 200 μl of a 1:1 mixture of ovalbumin and liposomal IMM60 (3rd dilution).

Animals were injected intravenously in the lateral vein of the tail. Cages containing 3 mice were heated in a heating chamber (37°C) for 20-30 minutes. Individual mice were placed in a restrainer that allowed the tail to extend out. Using a 27G insulin syringe needle, mice were injected with a 200 μl recording dose in approximately 4 seconds. Group sizes are detailed in Table 12 below, and mice were divided into groups of 4-6 and housed in individually ventilated cages:

Table 12

18 hours after the injection dose, blood samples were taken for serum analysis, and mice were euthanized and spleen cells were recovered to analyze the maturity of CD11c+ cells.

Blood Sampling - Serum: Warm cages containing 3 mice in a heating chamber at 37°C for 20-30 minutes, then place each mouse in a restrainer with its tail exposed. Make a nick in the lateral vein of the tail with a scalpel and collect blood into a serum separator tube. Stop blood flow by applying pressure and return the mouse to its normal cage. Allow blood to coagulate for 30 minutes at room temperature and then separate serum by centrifugation at 13,000 g for 2 minutes in a tabletop microcentrifuge. Transfer serum to wells of a 9U bottom plate and freeze at -20°C. Thawed samples are then used in standard sandwich ELISA without dilution.

Blood sampling - cells: Blood was collected from the lateral tail vein as described above, but instead of using a serum separator tube, a drop of blood (approximately 100 μl) was collected into a 1.5 ml microcentrifuge tube containing 200 μl of 10 mM EDTA/PBS solution and mixed by multiple inversions. The cells were kept on ice until the entire cage of mice had been bled. 1 ml of erythrocyte lysis buffer (Qiagen) was added to the blood and mixed by inversion. The samples were placed on ice until they became translucent. The tubes were then centrifuged at room temperature for 4 minutes in a microcentrifuge at 4K RPM. The supernatant was removed along with the plate, leaving a tan pellet. The cells in the pellet were washed with 200 μl of cold PBS and transferred to a U-bottom well of a 96-well plate and pelleted at 577g (1800 rpm) for 2 minutes. The supernatant was removed and the cells were treated with Fc Block (anti-CD16/CD32 antibody). The pellet was resuspended in 10 μl of tetrameric pMHC K ^b /SIINFEKL diluted 1:10 and the plate was covered with aluminum foil and incubated for 30 minutes at 37° C. The cells were washed once in cold PBS and resuspended in a cocktail of fluorescent antibodies, placed on ice for 15 minutes, then washed twice in PBS + 0.1% FBS, then diluted with 200 μl of the same solution and analyzed using a BDLSR Fortessa FACS machine.

Splenocytes: Mice were euthanized (cervical dislocation) according to the procedure in Appendix 1, and spleens were collected and stored in 1.5 ml of RPMI on ice. The spleen was crushed with the flat end of a 1 ml syringe plunger and squeezed through a 70 μm sieve into a 50 ml falcon tube and centrifuged at 1500 rpm for 5 minutes in an under-the-counter centrifuge (484 g) for granulation of cells. Splenocytes were resuspended in 2 ml of erythrocyte lysis buffer (Quiagen) and diluted with 8 ml of complete medium (RPMI, 10% FBS, Pen/strep, Glut) after treatment at room temperature for 2 minutes. Splenocytes were mixed and then granulated by centrifugation (484 g) at room temperature. Splenocytes were then resuspended in 3 ml of complete medium and counted. 2×1 splenocyte samples were placed in one well of a 96U bottom well plate. Cells were granulated by centrifugation at 1800 rpm (577 g) for 2 minutes, and the supernatant was discarded. Each pellet was resuspended in a mixture of fluorescent antibodies and placed on ice for 15 minutes, then washed twice with PBS + 0.1% FBS, diluted with 200 μl of the same solution, and analyzed using a BD LSR Fortessa FACS machine.

High protein binding (Maxisorb), 96-well flat bottom plates (Sigma) were coated with rat anti-mouse IFN-γ capture antibody clone R4-6A2 (5 μg/ml) diluted in sodium bicarbonate solution (0.05M and pH = 9.5). The night before, the plates were placed at 4°C overnight, washed 3 times in PBS-0.005% Tween-20, and blocked with 2.5% FBS in PBS for 2 hours. After washing again 3 times in PBS-0.005% Tween-20, serum samples and a series of repeated dilutions of standards ranging from 500ng/ml to 0.488ng/ml (recombinant mouse IFN-γ (Peprotech)) and repeated blanks (PBS) were added to the plates. The plates were placed at 4°C overnight.

The samples were then removed and washed 6 times, after which a detection antibody solution (biotinylated clone XMG1.2 (0.5 μg/ml)) was added and left at room temperature for 2 hours.

After washing three more times, add streptavidin-HRP solution (dilute the stock solution from Invitrogen 1000 times) and leave at room temperature for 1 hour. Wash the plate again six times with PBS-0.005% Tween-20, and then wash twice with PBS. Add TMB (sigma), cover with silver foil, leave at room temperature and check regularly. When the 250ng/ml standard reaches saturation (about 10-15 minutes), add an equal volume of 0.05N sulfuric acid to stop the colorimetric reaction. Use the SPECTROstar Nano disc reader to analyze the absorbance of each well at 450nm. The software of the SPECTROstar Nano disc reader converts the absorbance into ng/ml using the calibration curve generated by the standard dilution.

Injection of either IMM60 formulation (co-injected with ovalbumin) resulted in high levels of IFN-γ accumulation in the serum of treated mice 18 hours later (Figure 6). There was no statistically significant difference in the levels of IFN-γ induction between the two groups. As previously discussed (Jukes JP et al., J. Eur. Immunol. 2014 6: 1224-1234), non-glycosidic compounds can stimulate iNKT cells in humans and mice); the source of IFN-γ is NK cells that have been transactivated by iNKT cells to produce IFN-γ, and these cells produce the same cytokine at an earlier stage after recognizing iNKT cell agonists. In contrast, no induction of IFN-γ was observed in animals that were untreated or treated with vehicle (and ovalbumin) alone. Injection of liposomal IMM60 (co-injected with ovalbumin) produced moderate amounts of serum IFN-γ.

Maturation of CD11c+ dendritic cells in mice treated with liposomal IMM60 versus soluble IMM60: Dendritic cell maturity was determined by measuring the levels of the co-stimulatory molecule CD8 on the surface of CD11c+MHC class II+ cells. In this assay, a significant difference in the efficacy of IMM60 was observed when it was delivered as a soluble molecule compared to when it was delivered as a liposomal formulation (Figure 7). The same amount of IMM60 (0.1 ng/mouse) was used in both treatments, however, the liposomal formulation induced 2.5-fold higher levels of CD8 dendritic cells. The experiment also showed that at this time point, a 10-fold reduction in the dose of liposomal IMM60 did not mature dendritic cells above background levels. (Figure 7) This result is in stark contrast to the stimulatory effect of systemic IFN-γ production observed using this same dose (Figure 6).

Seven days after the mice were injected, blood samples were taken and blood cells were stained with fluorescent antibodies specific for lymphocyte markers and tetrameric pMHC:H-2K ^b containing the ovalbumin peptide SIINFEKL. Figure 8 shows the percentage of T cells that bound to tetrameric pMHC; on average, mice that received co-injections of ovalbumin with liposomal IMM60 (0.1 ng/mouse) produced significantly more antigen-specific T cells. This is consistent with the data shown in Figure 7, showing that CD11c+MHC class II+ dendritic cells were highly mature in this group of mice and that they would become better antigen-presenting cells than dendritic cells from any other group of mice.

Conclusion: The results of this experiment show that the liposomal formulation of IMM60 enhances the ability of IMM60 to stimulate antigen-specific immune responses, which is essential for the initiation of anti-tumor immune responses. Under these conditions, IFN-γ production may have reached a maximum level, but it is the maturation level of dendritic cells, rather than IFN-γ production, that is associated with the increase in antigen-specific T cell responses. The results also show that the maturation of dendritic cells is central to the conversion of early innate immune responses to adaptive immune responses and is effectively triggered by liposomal formulations of IMM60.

Example 4: Six different liposome formulations of IMM60 (POPC/DDAB, DSPC/DSPG and POPC/DC-Chol) were injected intravenously into mice

Fig. 5 shows the results of the experiment of measuring INF-γ after applying six different IMM60 prototype liposome preparations 18 hours compared with soluble αGalCer. The experiment shows that the DSPC/DSPG prototype liposome has the highest dynamic range (slow and elevated response at different doses) and the response at the lowest dose. The dosage is (from left to right) 0.01ng/mL, 0.1ng/mL, 1g/mL, 5ng/mL, and the injection volume is 100uL. The positive control used in this experiment is 1.0ng/mL of soluble α-GalCer. The data also show that for the second to fifth prototype liposomes, compared with the soluble α-GalCer of 1.0ng/mL, the IFNγ measured values of the two higher doses are greatly increased.

Example 5: Intravenous injection of three different liposome formulations IMM60 (POPC/DDAB, DSPC/DSPG and POPC/DC-Chol) into mice

Three different liposomal formulations of IMM60 (POPC/DDAB, DSPC/DSPG, and POPC/DC-Chol) were tested by intravenous injection in mice. The effects of the different liposomal formulations on the ability of IMM60 to induce IFN-γ levels in serum over 18 hours, as well as the extent of iNKT cell-mediated maturation of splenic dendritic cells (DCs) and compensatory upregulation of PD-L1 were determined (Figure 1A to Figure 1C). CD1d knockout mice were used as a control because CD1 knockout mice (due to lack of CD1 expression) do not generate iNKT cells.

Three IMM60 liposomal formulations, POPC/DDAB, DSPC/DSPG, and POPC/DC-Chol, were injected intravenously into wild-type C57BL/6 mice or CD1d KO mice, which lack both the presenting molecule DC1d and invariant natural killer T cells. After 18 hours, serum IFN-γ levels were measured, and splenic DCs were tested for surface expression of CD86, CD40, and PD-L1.

The effects of IMM60 treatment on IFN-γ induction and DC maturation proved to be target-dependent (CD1d) and independent of the liposomal formulation, as no effects were observed in CD1d knockout mice (Figures 1A to 1C). Dose-dependent induction of serum IFN-γ was observed with all three liposomal formulations within 18 hours. However, the POPC/DC-Chol formulation was significantly less potent in inducing IFN-γ than the POPC/DDAB or DSPC/DSPG (liposomal IMM60) formulations. (See Figure 1A). At the highest dose tested, liposomal IMM60 produced the broadest dose response, as well as the greatest increase in IFN-γ. All three liposomal formulations had a fairly strong effect on DC maturation, and this effect was already maximal at the lowest dose tested. A compensatory increase in the expression of the immunosuppressive PD-L1 ligand on the DC surface was observed (Figure 1C).

Example 6: Intravenous injection of three different liposome formulations IMM60 (POPC/DDAB, DSPC/DSPG and POPC/DC-Chol) into mice

Fig. 2 to Fig. 4 show the result of experiment, in this experiment, 0.01ng, 0.1ng and 1ng of POPC/DDAB, DSPC/DSPG or POPC/DC-Chol IMM60 prepared in the liposome composition disclosed herein was intravenously injected into wild-type C57BL/ mice. After injection 4 hours, 18 hours and 72 hours, the level of IFNγ, IL-12p70, IL-2, IL6 and IL-4 was measured. This experiment explored the dosage range lower than example 4, and increased additional cytokine output. When the IMM60 (soluble IMM60, not prepared in liposome) dissolved was injected as a positive comparison control and injected together with the vehicle negative control, the measured values of these liposomes were compared with the measured values of the same markers.

Example 7: In vivo stability of liposome IMM60 after intravenous injection into mice

Introduction

To estimate the biological half-life of IMM60 after intravenous injection of liposomal IMM60, two doses (0.5 μg/Kg and 50 μg/Kg) were injected into two strains of mice 10 minutes or 180 minutes after injection of liposomal IMM60, and the level of IMM60 in plasma was measured afterwards. The two strains of mice compared were CD1 mice (not to be confused with CD1d-KO mice) and C57BL/6 mice. Half-life, volume of distribution, clearance, AUC and _Cmax were calculated using a one-compartment model using first-order kinetics. The group mean plasma concentrations showed that there was a proportional dose response between the low dose (0.5 μg/kg) and the high dose (50 μg/kg) of the two mouse strains. In the two strains of mice, the values obtained in terms of half-life, volume of distribution and clearance were comparable. Values for soluble IMM60 injected mice were also obtained, in which a longer half-life of IMM60 was observed and a larger volume of distribution.

At a dose of 50 mg/kg, the estimated half-lives were 47.3 minutes in C57BL/6 and 52.4 minutes in CD1 mice, respectively. Half-lives could not be estimated for either strain of mouse at a dose of 0.5 mg/kg because exposures were below the limit of quantification at 180 minutes in low-dose animals.

Lipid formulation: DSPG: DSPC: IMM60 (3.44: 4.37: 1 mg/ml) in a buffer containing 250 mM sucrose, 145 mM sodium chloride and 10 mM sodium phosphate at pH 6.5. Prior to use, vials of liposome IMM60 were thawed at room temperature (21°C to 22°C) and then heated to 55°C in a heated water bath for 10 minutes. The vials were inverted six times for mixing, cooled to room temperature, and then diluted to 100 ng/ml or 10,000 ng/ml with injection grade saline (0.9% sodium chloride).

Soluble IMM60: Lyophilized material was resuspended to 10 mg/ml (10 μl per 100 μg vial) in chloroform: methanol: water (10:10:3), spun to capture all material, then vehicle solution (150 mM sodium chloride, 0.5% Tween20 in distilled water) was quickly added to a final concentration of 100 μg/ml (for 100 μg in 10 μl, 990 μl of vehicle was added), then heated to 80°C to 90°C for 5 minutes and sonicated for 5 minutes using a water bath sonicator. Samples were diluted to 10,000 ng/ml with injection grade saline.

The mice were placed in a cage in a 37°C heating chamber to pre-warm for 20-30 minutes. Each group of mice was injected intravenously with a 27G insulin syringe needle with a recorded dose of 100 μl (C57BL/6 mice) or 150 μl (CD-1 mice) over approximately 4 seconds.

Plasma samples were analyzed by LC/MS/MS at Frontage Laboratories. After administration of liposomal IMM60 injection, the assay for measuring IMM60 in plasma did not distinguish between liposome-bound IMM60 and soluble (unbound) forms of IMM60. The method used α-GalCer (an analog of IMM60) as an internal standard, and mass spectrometry ionization was electrospray in positive ionization mode. The range of calibration standards in mouse plasma was 1 ng/mL to 1000 ng/mL of IMM60.

10 minutes and 180 minutes after IV administration, plasma concentration is measured. Half-life, clearance and AUC _(0→∞) are calculated using a one-compartment model using first-order kinetics, because only two time points along the PK curve are samples. PK parameters can be calculated using these assumptions. Other assumptions are to replace T ₀ with T _{10 minutes} and to replace C ₀ and C _maxima with C _{10 minutes} to solve the PK parameters of liposome IMM60.

The half-life of IMM60 was calculated as (1) t _1/2 = 0.693/ _ke , where _ke = (ln C _{10 min} - ln C _{180 min} )/(180 _min - 10 _min ).

(2) V _d = dose/C _{10 min} , where dose is expressed as original or dose/body weight

(3) Cl = _ke × V _d = (ln C _{10 minutes} - ln C _{180 minutes} ) / (180 _minutes - 10 _minutes ) × V _d

AUC calculation of IMM60 (4) AUC _(0→∞) = C _10min / _ke

Statistical calculations: Mean, standard deviation (SD), standard error of the mean (SE), p-values for t-tests (equal or unequal variances) were calculated using Microsoft Excel 201 (Microsoft Corporation). Equal or unequal variances were selected using the F-test function. All t-test comparisons were performed using 1000 ng of liposomal IMM60 as a control. Half-life of IMM60 in mice T(1/2): The half-life of IMM60 in mice was calculated using Equation 1 using a one-compartment model with first-order kinetics.

Table 13. Half-life of IMM60 in mice

Volume of distribution: The volume of distribution of IMM60 in mice was calculated using Equation 2 using a one-compartment model with first-order kinetics.

Table 14. Distribution volume V(D) of IMM60 in mice

Elimination of IMM60: The clearance of IMM60 in mice was calculated using Equation 3 using a one-compartment model with first-order kinetics.

Table 15. IMM60 clearance in mice

Exposure (AUC _(0-∞) ) of IMM60: The exposure (AUC _(0-∞) ) of IMM60 in mice was calculated using Equation 4 using a one-compartment model with first-order kinetics.

Table 16. Exposure (AUC _(0-∞) ) of IMM60 in mice.

_Cmax of IMM60: _The Cmax of IMM60 in mice was calculated using the T time point concentration of _{10 min} .

Table 17. Cmax _values of IMM60 in mice

Pharmacokinetic parameters of high-dose (50 μg/kg) liposome and soluble IMM60 injection in mice.

The PK summary of IMM60 in mice was calculated using the above formula and using a one-compartment model with first-order kinetics.

Table 18. Pharmacokinetic parameters of high-dose liposome IMM60 injection in mice

analyze

All comparisons were made assuming that the high-dose C57BL/6 strain was used as a reference for comparison.

Half-life: The half-life of the liposomal IMM60 formulation in the C57BL/6 and CD1 strains was considered equivalent, with a T _1/2 between 47 and 52 minutes at high doses. Since the 3-hour plasma samples were below the LOQ of the assay, the low dose could not be calculated. The half-life of the soluble formulation appeared to be longer with a T _1/2 between 95 and 139 minutes at high doses.

Volume of distribution: The volume of distribution of the liposomal IMM60 formulation was found to be equivalent in the C57BL/6 and CD1 strains, with _Vd ranging from 303 ml/kg to 383 ml/kg at high doses. Low doses in each strain were also found to be equivalent, with _Vd ranging from 262 ml/kg to 263 ml/kg. The volume of distribution of the soluble formulation appeared to be greater, with _Vd ranging from 558 ml/kg to 639 ml/kg.

Clearance: The clearance of the liposomal IMM60 formulation was found to be equivalent in the C57BL/6 and CD1 strains, with clearance ranging from 4.3 ml/kg/min to 5 ml/kg/min at high doses. Low doses could not be calculated as the 3 hour plasma samples were below the LOQ of the assay. The clearance of the soluble formulation appeared to be lower, with values ranging from 3.1 ml/kg/min to 4.1 ml/kg/min.

Exposure: Liposomal IMM60 formulations were found to have equivalent exposure in C57BL/6 and CD1 strains, with AUC _(0-∞) ranging from 9,030 ng/mL/min to 15,104 ng/mL/min at high doses. Low doses could not be calculated as 3-hour plasma samples were below the assay LOQ. Exposure with the soluble formulation appeared to be greater at high doses, with AUC _(0-∞) ranging from 12,225 ng/ml/min to 16,165 ng/ml/min.

_Cmax : The Cmax _values of the liposomal IMM60 formulations in the C57BL/6 and CD1 strains were considered equivalent, with concentrations ranging from 134 ng/mL to 190 ng/mL at the high dose. When dose matching was performed using the IMM60 dose (μg/kg) of the low-dose and high-dose animals, the low dose in each strain was also considered equivalent (the low dose was 100-fold lower than the high dose). The Cmax _values of the low dose were approximately 2.1 ng/mL for both mouse strains. The Cmax _values of the soluble formulation (high dose) appeared to be lower, with concentrations ranging from 84 ng/mL to 90 ng/mL. The greater Cmax _values of the liposomal formulations are considered advantageous and a sign of improved efficacy of the liposomal formulation. In some embodiments, the invention includes liposomal formulations of the API that, when administered to C57BL/6 or CD1 mice, provide _{a Cmax} that is at least 20% greater, or at least 30% greater, or at least 40% greater, or at least 50% greater, or at least 75% greater, or at least 100% greater than the Cmax observed with an equivalent dose of the soluble API.

No pharmacokinetic differences were detected between the C57BL/6 and CD1 mouse strains. No PK differences in volume of distribution and dose- _proportional Cmax were detected between high or low doses. Since the 3-hour plasma samples were below the assay LOQ, half-life, clearance, and exposure could not be calculated. At high doses, pharmacokinetic differences were detected between the liposomal and soluble formulations in T _1/2 , V _d , clearance, exposure, and _Cmax .

The lower distribution volume ( _Vd ) of the liposomal formulation is advantageous, indicating that the concentration of IMM60 in the plasma of the liposomal formulation is 32% to 53% higher than that of the soluble formulation after injection. The lower Vd indicates that there are fewer IMM60 partitions outside the plasma (blood) cavity. Since more IMM60 enters the blood immediately after injection, more IMM60 can penetrate into the tumor through tumor vascular extravasation. Due to the lower Vd, liposomes are more effective than soluble IMM60 and have less toxicity. In some embodiments, the present invention includes a liposomal formulation of API, and when the liposomal formulation is administered to C57BL/6 or CD1 mice, the _Vd provided (measured in (mL/kg)) is at least 20% smaller than the _Vd observed with a soluble API dose of the same size dose, or at least 30% smaller, or at least 40% smaller, or at least 50% smaller.

Example 8: Injection of liposome IMM60 into mouse melanoma

To evaluate the anticancer therapeutic properties of liposomal IMM60, the potential of liposomal IMM60 to prevent lung metastatic lesions in a mouse melanoma model was tested in both preventive and therapeutic settings. Three doses of liposomal IMM60 were injected intravenously 3 days before (prevention) or 3 days after (treatment) tumor cell injection. Mice were injected intravenously with B16-F10 melanoma cells, and metastatic nodules in the lungs were counted 15 days later.

Prophylactic treatment was more effective than therapeutic treatment in preventing lung tumor formation; however, in both prophylactic and therapeutic treatments, even at the lowest liposomal IMM60 dose (0.01 ng/mouse), tumors were significantly reduced compared to mock-treated mice (Figures 9A and 9B). In fact, the greatest effect was found for 0.01 ng liposomal IMM60/mouse, with no significant additional benefit found at larger liposomal IMM60 doses. Treatment of mice with liposomal IMM60 (DSPC/DSPG-IMM60) at 1 of 3 doses (0.01 ng, 0.1 ng, or 1 ng per mouse) 3 days before tumor administration (prophylactic setting; Figure 9A) or 3 days after tumor administration (therapeutic setting; Figure 9B) reduced the number of metastatic nodules in the lungs of mice.

Example 9: Combination therapy with anti-PD-1 blocking antigen - B1 tumor model

Treatment with soluble IMM60 upregulated PD-1 levels on iNKT cells. The same treatment also upregulated the expression of PD-1's ligand, PD-L1, on various immune cells. (See, e.g., Figure 1C and Figure 1F [liposomal IMM60]). We investigated the possibility that combination therapy using liposomal IMM60 and anti-PD-1 blocking antibodies could be used to enhance inhibition of tumor growth.

The combination therapy was evaluated in a therapeutic setting using an unresponsive B16-F10 mouse melanoma SC model (“cold tumor model”), with treatments given on days 3, 6, and 9 after subcutaneous tumor implantation. Two doses of liposomal IMM60 were tested together with an immune checkpoint inhibitor anti-PD-1 antibody (10 mg/kg) and compared to liposomal IMM60 alone, anti-PD-1 antibody alone, or a control antibody of the same type.

IFN-γ was measured in blood samples 18 hours after treatment with liposomal IMM60. As shown in Figure 10, the activity of liposomal IMM60 was proportional to the dose, with the high dose (10 ng/mouse) producing more IFN-γ than the low dose (0.1 ng/mouse). Treatment with anti-PD-1 antibody alone had no effect on serum IFN-γ. Importantly, the combination of anti-PD-1 antibody with a 10 ng dose/mouse of liposomal IMM60 produced higher levels of IFN-γ compared to liposomal IMM60 treatment alone.

As expected, anti-PD-1 treatment alone had no effect on the growth of B16-F10 tumors, while single-agent liposomal IMM60, either at a low dose (0.1 ng/mouse) or a high dose (10 ng/mouse), had only a modest antitumor effect. However, the combination of anti-PD-1 and a high dose of liposomal IMM60 (10 ng/mouse) significantly reduced tumor growth (as indicated by the arrows in Figure 11).

Example 10: Combination therapy with anti-PD-1 blocking antigen - CT-2 tumor model

Overview

The effects of IMM60 liposomes as a single treatment or in combination with anti-mouse PD-1 on tumor growth and survival in the CT-2 tumor model in BALB/c mice were evaluated, and IFN-γ levels in the serum of experimental mice were measured 18 hours after the first treatment.

Tumor volumes were measured in mice over 14 days. The interaction showed a statistically significant trend in the fold change over time, and the effect of treatment was also close to a significant trend. Post hoc analysis detected significant differences between the IMM60/anti-PD1 group and the vehicle control group on days 11 and 14.

There were no significant differences in the survival curves due to treatment.

Serum IFN-γ levels were significantly increased in the IMM60 and IMM60/anti-PD1 treatment groups relative to the anti-PD1 group or vehicle control group. Combination of IMM60 and anti-PD1 resulted in approximately 2-fold higher IFN-γ levels compared to IMM60 treatment alone.

Experimental details and results

Body weight and tumor measurements: Animal body weights were measured on days -1, 0, 2, 4, 7, 9, 11, 14, 16 and 18. Tumors were measured on study days 0, 2, 4, 7, 9, 11, 14, 16 and 18. Tumors were measured in two dimensions using calipers. Tumor volume was calculated using the formula: Volume ( ^mm3 ) = (length x ^width2 )/2.

64 mice were subcutaneously inoculated with 0.3×10 CT-2 cells. On days 0 and 10 after inoculation of CT-2 cells, when the average tumor volume reached approximately 100 mm ³ , 32 mice were grouped and treated. The four treatment groups received vehicle, IMM60, anti-PD1, or a combination of IMM60 and anti-PD1, as shown in the table below.

Experimental design of the main study

IV - intravenous injection IP - intraperitoneal injection

Serum IFN-γ levels: On day 1, 18 hours after the first treatment, each group of mice was bled through the retro-orbital sinus under isoflurane anesthesia. The whole blood was coagulated for 30 minutes at room temperature, centrifuged at 1000 x g for 15 minutes, and serum was separated from the whole blood and frozen at -80°C until analysis. IFN-γ levels were measured using Meso Scale Discovery (MSD) according to the manufacturer's protocol.

During the study, the body weights of the animals in the different groups were closely followed.

Tumor volume measurement data are depicted in Figure 12A. When these data were analyzed by two-way repeated measures ANOVA, the interaction showed a statistically significant trend (P = 0.0698), and the treatment effect was also close to a significant trend (P = 0.1142). Bonferroni's post hoc analysis is shown in Figure 12B, and significant differences were detected between the IMM60/anti-PD1 group and the vehicle control group on days 11 and 14.

Tumor volumes for individual animals are shown in Figure 12C. One-way ANOVA of these data did not reveal significant differences due to treatment (P = 0.3423).

Figure 12D shows the IFN-γ levels measured at 18 hours. A significant effect of treatment was detected in a one-way ANOVA (P<0.0001). Relative to the anti-PD1 group or vehicle control, the IFN-γ levels in the IMM60 and IMM60/anti-PD1 treatment groups were significantly increased. The statistical differences between the treatment groups and the vehicle control group are shown in the figure. Tukey post hoc analysis comparisons for all groups are shown below the chart. Compared with treatment with IMM60 alone, the combination of IMM60 and anti-PD1 increased the IFN-γ levels by about 2 times.

These data from Examples 9 and 10 demonstrate that, in the setting of limited IFN-γ responses to iNKT cell stimulation, the addition of complimentary immune checkpoint inhibition therapy augments responses in tumors that were unresponsive to either therapy alone.

Example 11 - IMM60 induces PD-L1 expression on melanoma cells co-cultured with mouse spleen cells

Overview

Using an in vitro mouse cell model, we measured the effect of IMM60 on PD-L1 expression levels on the surface of tumor cells co-cultured with splenocytes. More specifically, we examined PD-L1 expression on the mouse melanoma tumor cell line B16f10 co-cultured with splenocytes (as a source of iNKT cells and antigen presenting cells (APCs), such as CD1 ⁺ B cells and CD11c ⁺ dendritic cells, with approximately 1% of viable cells present in splenocytes in vitro).

Splenocytes from wild-type mice were treated with IMM60, IMM47, or vehicle and anti-PD-1 or isotype control antibodies. 48 hours later, PD-L1 levels on the surface of B16f10 cells were determined by antibody labeling and flow cytometry.

Addition of IMM60 resulted in upregulation of surface PD-L1 in the mouse melanoma cell line B16f10, whereas addition of the less potent iNKT agonist IMM47 did not upregulate surface PD-L1.

We also evaluated the effect of checkpoint inhibition on this upregulation of PD-L1 expression. Co-administration of an anti-PD-1 antibody with IMM60 increased PD-L1 levels on melanoma cells; no such enhancement was observed on T cells.

Materials and methods

Preparation of reagents and materials:

IMM60 and IMM47 were lyophilized and stored at -20°C.

Soluble IMM60 or IMM47: The lyophilized material was resuspended in chloroform: methanol: water (10: 10: 3) to 10 mg / ml (10 μl vial containing 100 μg), spun to capture all the material, and then the vehicle solution (150mM sodium chloride, 0.5% Tween20 in distilled water) was quickly added to a final concentration of 100 μg / ml (for 100 μg in 10 μl, 990 μl of vehicle was added). The solution was then heated to 80 ° C for 5 minutes and sonicated for 5 minutes using a water bath sonicator. After reconstitution in glass vials, both IMM60 and IMM47 were stored at 4 ° C, and they were sonicated for 5 minutes in a water bath sonicator before each use.

The antibodies used were anti-PD-1 antibody (rat anti-mouse PD-1) clone RMP1-14 (IgG2a, k) from BioXCell InVivoPlus and isotype control antibody (rat IgG2a, k) clone 2A3 from BioXCell InVivoPlus. Before use, the antibodies were diluted to the desired concentration with complete medium (RPMI, 10% FBS, Pen/Strep and glutamine).

Two 8-week-old C57BL/6J OlaHsd female mice were used in this study. The mice were purchased from Envigo and weighed 19 gr to 20 gr. One male mutant mouse (Ja18 KO mutation, preventing the expression of constant natural killer T cell receptor (NKT TCR)) was used, which was from a colony maintained at the Oxford University Biomedical Animal Facility.

For fluorescence activated cell sorting (FACS), the following materials were used: phosphate buffered saline (PBS), RPMI, complete medium: (RPMI with 10% bovine serum (FBS), penicillin (25,000U), streptomycin (25mg/L), glutamine (2.1mM), (all tissue culture grade products were from Gibco); erythrocyte lysis buffer (Qiagen). All antibodies were purchased from eBioscience or Biolegend: CD11b-FITC, CD105-BV421, CD45.2-APC, TCRpanBeta-APC-cy7, PD-L1-PE, CD69-PEcy7, (from Biolegend), and live/dead fixative stain (Thermo Fisher Scientific). Fc blocking antibody anti-CD16/CD32 (eBioscience).

Anti-PD-1 (RMPI-14) and isotype control (2A3) were both diluted from stock solutions to 400 μg/ml. The antibodies were then further diluted 1:4 to 100 μg/ml, which was the final concentration in the wells.

To obtain splenocytes, mice were euthanized by cervical dislocation, and spleens were collected and frozen in 1.5 ml RPMI. The spleen was crushed with the flat end of a 1 ml syringe plunger and squeezed through a 70 μm sieve into a 50 ml Falcon tube, and the cells were granulated by centrifugation at 1500 rpm for 5 minutes in an under-the-counter centrifuge (484 g). Splenocytes were resuspended in 2 ml of erythrocyte lysis buffer (Qiagen), and diluted with 8 ml of complete medium (RPMI, 10% FBS, Pen/strep, Glut) after 2 minutes at room temperature. Splenocytes were washed and then granulated by centrifugation (484 g) at room temperature. Splenocytes were then resuspended in 3 ml of complete medium and counted. Cells were resuspended at 5 × 1e7/ml, and 100 μl (5 × 1e6) of splenocytes were aliquoted in a well of a 96U bottom well plate.

Determination

100 μl of spleen cells were placed in a well of a 96U bottom well plate. Antibodies RMPI-14 and 2A3 were diluted to 4 times the final concentration (400 μg/ml) and 50 μl was added to each well to achieve a final concentration of 100 μg/ml.

Before adding iNKT cell agonists, cells were incubated for 10 min at room temperature to allow antibody binding.

An additional 50 μl of complete medium was added, wherein the concentration of complete medium was the final concentration of IMM60, IMM47 or vehicle × 4. The final effective concentrations of IMM60 or IMM47 were determined to be 0.0, 0.0001, 0.001, 0.01, 0.1, 1.1, 11 and 110 ng/ml.

The plates were moved to a 37°C, 5% _CO2 incubator for 48 hours. The plates were then removed, washed, treated with purified anti-mouse CD16/CD32 (1:50 dilution Biolegend), then washed, and stained with fluorescent antibodies and vital staining as summarized in the following table:

FACS analysis of splenocytes.

The cells were pelleted by centrifugation at 1800 rpm (577 g) for 2 minutes and the supernatant was discarded. Each pellet was resuspended in a mixture of fluorescent antibodies and placed on ice for 20 minutes, then washed twice with PBS + 0.1% FBS, diluted with 200 μl of the same solution, and analyzed using a BD LSR Fortessa FACS machine. Finally, the data were analyzed using FlowJo software (CricketGraph).

For B16f10, live cells were gated, followed by CD45.2-negative (non-immune cells) CD105-positive (aB16f10 marker). A final gate was set to focus on the main B16f10 population based on forward and side scatter (indicators of size and granularity, respectively). Finally, the final gated population was evaluated for PD-L1 levels. FACS analysis data of splenocytes were captured using BD LSRFortessa and then analyzed using FlowJo software. Finally, GraphPad's Prism statistical analysis software was used to plot and analyze the data.

result

PD-L1 levels on B16f10 cells increased with treatment with higher concentrations of IMM60.

Increasing concentrations of IMM60 led to increased expression of PD-L1 on B16f10 cells when B16f10 was co-cultured with splenocytes from C57BL/6 mice. This increase was not observed when vehicle alone or IMM60 was added to B16f10 co-cultured with splenocytes from Ja18 KO mice (which did not contain any iNKT cells).

(Figure 13)

Combination treatment with IMM60 and anti-PD-1 further increased surface PD-L1 levels.

In a parallel set of experiments, co-cultured B16f10 cells and splenocytes were incubated with different concentrations of IMM60 and IMM47 and supplemented with antibodies (100 μg/ml), either anti-mouse PD-1 (clone RMPI-14) or isotype control antibody (clone 2A3). Figure 13 shows that PD-L1 expression was slightly increased when anti-PD-1 antibody was used compared to isotype control antibody. This increase was only seen in co-cultures at and above 11 ng/ml IMM60 (indicated by arrows).

T cells (identified using TCRpanBeta antibody) in the same B16f10 cell:splenocyte co-culture also upregulated surface PD-L1 when IMM60 was added. However, unlike B16f10 cells, this upregulation was not affected by the presence of anti-PD-1 antibody (Figure 14).

in conclusion

The results of this experiment show that splenocytes co-cultured in vitro contain sufficient numbers of iNKT cells to produce agonist-specific effects on B16f10 melanoma cells co-cultured with them. This effect may be somewhat weak compared to the effects that can be seen in vivo. In vivo, we have observed IFN-γ responses using as little as 0.01 ng/mouse when injected intravenously. See Jukes JP et al. J. Eur. Immunol. (2016) 46: 1224-1234.

Non-glycoside compounds can stimulate human and mouse iNKT cells. This sensitivity is likely due to the capture efficiency of IMM60 in live mice and the efficient delivery to iNKT cells compared to in vitro assays. The efficiency of this capture and delivery has been demonstrated in a paper discussing the capture of blood-borne antigens by CD169+ marginal zone macrophages and dendritic cells, which can provide lipids to iNKT cells. See Barral et al. EMBO J. (2012). 31: 2378-2390. The location of spleen NKT cells facilitates their rapid activation by blood-borne antigens.

The results of this study also showed that IMM60-induced upregulation of PD-L1 on the surface of B16f10 cells was further enhanced after treatment with anti-PD-1 antibodies.

Surface PD-L1 is known to be upregulated in response to the presence of IFN-γ and thus may be a phenotypic surrogate for the presence of IFN-γ. However, the involvement of PD-L1 in the inhibition of PD-1+ T cells and the effects of blocking this interaction observed in this study suggest that the negative feedback loop may be disrupted by anti-PD1 antibodies, although the data from this study cannot determine whether it is PD-L1 on B16f10 cells that causes this negative feedback.

On T cells, upregulation of surface PD-L1 was evident after addition of IMM60 but was not enhanced by anti-PD1 antibody treatment. Therefore, disruption of the PD-1:PD-L1 axis and its effect on further upregulation of PD-L1 may be very local, with perhaps cognate and bystander cells such as conventional T cells being unaffected.

Example 12: Clinical trial protocol

The following is a clinical trial protocol for IMM60 administered alone or as part of a combination therapy. The dosages and dosing regimens described herein are considered specific examples. The protocol may be repeated/modified with different liposomal formulations of IMM60; it may also be repeated/modified with alternative secondary treatments such as different anti-PD-1 antibodies or different immune checkpoint inhibitors.

IMM60 and anti-PD1

Treatment of mice with IMM60 combined with anti-PD1 resulted in a dose-dependent increase in IFNγ secretion compared to IMM60 alone. This led to further activation of NK cells and increased IFN-γ secretion. In addition, anti-PD1 antibodies blocked PD1 expressed on the surface of iNKT cells, resulting in improved maturation of dendritic cells and B cells and promoted priming/enhancement of antigen-specific CD8+ T cells. In animals resistant to anti-PD1 treatment alone, the combination of IMM60 plus anti-PD1 antibodies restored sensitivity.

Medication supply and dispensing

Pembrolizumab is an anti-PD-1 antibody marketed by Merck under the trade name Pembrolizumab Infusion Solution is a sterile, non-pyrogenic aqueous solution supplied in single-use Type I glass vials containing 100 mg/4 ml of pembrolizumab. Pembrolizumab Infusion Solution should be prepared by the on-site pharmacy in 0.9% sodium chloride (normal saline) and the final concentration of pembrolizumab in the infusion solution should be between 1 mg/ml and 10 mg/ml. For intravesical infusion, the final instilled infusion volume will be 40 ml.

In some embodiments, IMM60 is formulated in liposomes as described herein. IMM60 should be stored frozen and then thawed and diluted by the on-site pharmacy before dispensing to the ward. The recommended dose of liposomal IMM60 is 1, 3 or 9 mg/m ² , intravenously infused in 250 ml of Macopharma saline diluent for 60 minutes, once every 3 weeks. IMM60 is supplied in the form of a 1 mg/ml solution, each vial contains 2 ml of IMM60, and the vial should be stored at -20°C, so the vial needs to be thawed in the pharmacy before dispensing. Before use, the vial should be thawed for 1 to 2 hours until it reaches ambient temperature. Heat the vial in a water bath at 55°C ± 2°C for 10 minutes. Invert and rotate the vial by hand several times and let it cool to room temperature before use.

Rationale for the study

This clinical trial provides a protocol to demonstrate the synergistic effect of IMM60 and pembrolizumab, which is licensed as standard of care, compared to pembrolizumab alone in patients with melanoma and non-small cell lung cancer. The trial evaluates whether IMM60 can restore sensitivity to pembrolizumab-resistant melanoma patients and determines whether IMM60 can promote PDL1 expression in PDL1-non-expressing small cell lung cancer. The clinical trial consists of two phases, Phase 1 and Phase 2. The clinical trial was conducted according to the protocol in Table CT-1.

Initial safety will be evaluated in multiple ascending dose cohorts of IMM60 followed by IMM60 + pembrolizumab.

Table CT-1

Table CT-2 below summarizes the timeline of events in the trial.

Rationality of medication

According to ICH S9 "Nonclinical Evaluation of Anticancer Drugs", the clinical starting dose will be set at the human equivalent dose (HED), which is 1/10 of the rodent STD10 or 1/6 of the highest non-rodent non-severe toxic dose (HNSTD), whichever is lower. For liposomal IMM60, the mouse STD10 is >5mg/kg (15mg/m ² ) because there were no deaths, clinical symptoms, or weight loss at this dose. Humans are expected to be much less sensitive than mice to the toxicological effects of liposomal IMM60 because these effects may be mediated by exaggerated pharmacological effects (excessive production of IFN-γ). Therefore, based on nonclinical safety studies in mice, the conservatively estimated clinical starting dose is 1/10 of the mouse STD10 or 1.5mg/m ² . The actual planned clinical dose was set at 1.0mg/m ² , which is lower than 1/10 of the mouse STD10 (1.5mg/m ² ).

Experimental Design

This will be a randomized Phase 1/2 trial. Initial safety will be evaluated in multiple ascending dose cohorts of IMM60, followed by IMM60 + pembrolizumab. The Trial Management Group (TMG) will review the Phase 1 data, current standard of care, and feasibility to make recommendations regarding Phase 2 cohorts. Planned arming of the Phase 2 component includes: pembrolizumab alone, and IMM60 + pembrolizumab for patients with NCSLC; and pembrolizumab alone, IMM60 + pembrolizumab, and additional IMM60 alone cohorts for patients with melanoma.

The Phase 1 trial is a modified 3+3 trial design. Three dose levels of IMM60 (1/3/9 mg/m ² ) were evaluated in the IMM60 ascending dose safety cohort. If one of the first three patients developed a predefined dose-limiting toxicity (DLT), the cohort was expanded to include all patients. If any additional patients in this cohort developed a DLT, this dose level was defined as the maximum tolerated dose (MTD). If more than 2 patients in the expanded cohort developed a DLT, the MTD was defined as a dose level lower than that of the cohort. If more than 2 patients in the lowest IMM60 dose level cohort developed a DLT, a reduction in this dose level was considered. If no patient in the highest dose cohort (9 mg/m ² ) developed a DLT, this cohort was still expanded to include all patients. To decide whether the next cohort can be opened at a higher dose level in Phase 1 of the study, the Trial Management Group (TMG) will review the available data (e.g., safety profile) once all participants in the previous cohort have completed the first cycle to Day 21. A subsequent safety review by the TMG will be performed prior to enrollment of patients in the new cohort.

For the IMM60 + pembrolizumab safety cohort, pembrolizumab will be administered in combination with IMM60. The dose of pembrolizumab will be administered as standard of care; the dose of IMM60 will be determined by the IMM60 dose-escalation cohort. This combined safety cohort will also use a modified 3+3 design—the starting dose level of IMM60 will be below the MTD of IMM60 (referred to as MTD-1), and the dose will be escalated to the cohort where IMM60 is at the MTD. For example, if the IMM60 MTD is 9 mg/m ² , the first combined cohort will use IMM60 at 3 mg/m ² and will escalate to the combined cohort using IMM60 at 9 mg/m ^2. If the IMM60 MTD is 1 mg/m ² (the lowest dose level in the escalating dose cohort), then the TMG will consider whether to start the combined safety cohort at the MTD or at a stepped-down dose. The DLT rules that apply to the IMM60 dose-escalation cohort will also apply to the combined safety cohort.

In the second phase of the trial, melanoma patients will be randomized in a 1:2 ratio to treatment arms: pembrolizumab according to its licensed indication or the combination of pembrolizumab + IMM60. An additional prospective cohort is offering IMM60 at the MTD every 3 weeks for patients who have failed pembrolizumab. Potential PDL1-positive NSCLC patients will be randomized in a 1:2 ratio to receive pembrolizumab according to its licensed indication or the combination of pembrolizumab + IMM60. Based on a review of IMM60 monotherapy activity, melanoma and NSCLC patients in the pembrolizumab monotherapy cohort may receive pembrolizumab + IMM60 if they do not respond on a 3-month CT scan.

Another cohort consisting of PDL1-NSCLC will receive one cycle of IMM60, with tumor biopsies performed before and after to identify any changes in PDL1 expression. After this cycle, patients will receive the combination of IMM60 + pembrolizumab and undergo a second biopsy. Based on the review of IMM60 monotherapy activity, a potential group of patients receiving pembrolizumab monotherapy will be allowed to receive IMM60 in combination with pembrolizumab at disease progression to see if sensitivity can be restored.

Dose escalation procedure

Evaluable patients are those who have received the Cycle 1 dose and completed the minimum safety assessment requirements within 21 days, or those who experience a DLT during the 21-day DLT assessment period. A dose escalation decision will be made when 3 evaluable patients in a cohort complete the 21-day DLT assessment period; if a cohort needs to be expanded to 6 patients due to DLT, a dose escalation decision will be made once the evaluable patients have completed the 21-day DLT period. Dose escalation will be based on toxicity information obtained from evaluable patients in the cohort during the initial 21-day treatment cycle. The dose escalation guidelines for the Phase 1 cohort are shown in Table CT-3 below.

Table CT-3

Start of Phase 2 program

The interim analysis will be performed in the Phase 1 cohort and once all patients in the Phase 1 cohort have completed their DLT period, the interim analysis will be reviewed by the TMG to determine if the trial will advance to Phase 2 and to determine which cohorts will be open. This analysis will be based solely on safety data and will be reviewed for the most severe toxicity. The following stopping rules will be applied; if any of the following occurs, then the trial will not advance to Phase 2: if the rate of DLTs exceeds 33% or the rate of treatment-related serious adverse events (SAEs) exceeds 50%. The TMG will review pharmacodynamic data with tissue and blood biomarkers, as well as efficacy data, particularly evidence of disease stabilization and immune activation.

Duration of Patient Participation: Patients receiving IMM60 may receive up to 6 cycles, with dosing every 3 weeks, and patients will attend a post-treatment visit 1 month after the last dose. Patients receiving pembrolizumab will remain in the study for a maximum of 1 year and 1 month from the first trial dose to the last protocol visit. The duration a patient remains in the trial depends on their treatment.

Post-Trial Care and Follow-up: Following the post-treatment visit, patients will receive standard of care. Patients will be followed for progression-free and overall survival data by their healthcare team during the follow-up period (up to 1 year) or until disease progression or death, whichever comes first.

The objectives and endpoints of this clinical trial are shown in Table CT-4.

Table CT-4

Reaction Assessment

Response Evaluation Criteria in Solid Tumors (RECIST): All patients must have at least 1 lesion, measurable using RECIST1.1, in an area not previously treated with radiation therapy.

Tumor Assessments: Radiographic assessments for malignancies will be performed per protocol prior to initiation of study treatment. These lesions will be followed throughout the study using the same methods used to detect lesions at baseline. To ensure compatibility, radiographic assessments used to assess response will be performed using the same techniques. When both methods are used to assess the anti-tumor effect of treatment, imaging-based assessments are superior to those performed by clinical examination.

Baseline assessment: This will include radiographic measurement of the extent of disease via CT scan or MRI scan (if applicable). All areas of disease present must be mentioned (even if the response of a specific lesion will not be tracked), and measurements of all measurable lesions must be recorded on the scan report. Any non-measurable lesions must be stated as present.

On-Treatment and Off-Study Assessments: Tumor assessments will be repeated on the schedule given herein or more frequently if clinically indicated. All lesions measured at baseline must be measured at subsequent disease assessments and recorded on the scan report. All non-measurable lesions noted at baseline must be reported as present or absent.

Radiographic assessment of malignancies must be performed per protocol prior to initiation of study treatment. These lesions will be followed throughout the study using the same method used to detect them at baseline. To ensure compatibility, radiographic assessments used to assess response must be performed using the same technique. When both methods are used to assess the antitumor effect of treatment, imaging-based assessments are superior to those performed by clinical examination.

The principal investigator must ensure that the radiologist is aware of the requirement to follow up and measure each target lesion mentioned at baseline and to comment on non-target lesions according to RECIST criteria. Patients who continue to receive pembrolizumab under NHS licence will be monitored for 1 year or until disease progression or death, whichever comes first. During this period, disease assessment will not require the use of RECIST and routine reporting will suffice.

Tumor Response: To determine complete response (CR) or partial response (PR) status, changes in tumor measurements must be confirmed by two consecutive observations, at least 4 weeks apart. To determine stable disease (SD) status, follow-up measurements must meet SD criteria at least once and at least six weeks after the start of study treatment. If rapid tumor progression occurs before completing 9 weeks of treatment, the patient will be classified as early progression (EP).

Tumor response should be classified as “not evaluable” (NE) only if it cannot be assigned to another response category, such as when baseline and/or follow-up assessments were not performed or were not appropriately performed.

The applicable overall response category for each visit that includes a disease assessment must be documented in the medical record for inclusion in the appropriate CRF in OpenClinica.

Best overall efficacy was defined as RECIST 1.1 response rate, including confirmed CR and PR.

Example 13: Alternative clinical trial protocols

Referring to Example 12 above, the following is another clinical trial protocol if IMM60 is administered alone or as part of a combination therapy. The dosages and dosing regimens described herein are considered specific embodiments. The protocol can be repeated/modified with different liposomal formulations of IMM60; it can also be repeated/modified with alternative secondary treatments such as different anti-PD-1 antibodies or different immune checkpoint inhibitors.

Study intervention and intervention format:

IMM60 Liposomal: 1/3/9 mg/m ² intravenously (IV) every 3 weeks (Q3W) for several cycles; each participant's dose will be calculated based on body surface area at baseline and recalculated if the participant's weight changes ≥ 10% during the study. In some variations, each patient is administered a fourth dose level of IMM60 of 24 mg, 27 mg, 30 mg, 33 mg, or 3 mg for one cycle.

Pembrolizumab (MK-3475): 200 mg IV every 3 weeks for up to 35 cycles or approximately 2 years

Condition/Disease/Target Group:

Phase 1 (see Figure 15A):

NSCLC-Stage IV NSCLC

-IMM60 Dose-escalation Group: Progression after systemic therapy consisting of at least platinum-containing chemotherapy and immunotherapy (sequentially or in combination).

-IMM60+Pembrolizumab dose safety grouping: Prior chemotherapy and/or immunotherapy is allowed

Melanoma – unresectable stage III or IV cutaneous or unknown primary melanoma

-IMM60 dose escalation cohorts: Patients who have progressed on at least one line of immunotherapy. For those with BRAF-mutated tumors who have progressed on BRAF/mitogen-activated protein kinase inhibitor therapy and at least one line of immunotherapy.

-IMM60+Pembrolizumab Dose Safety Groups: Prior chemotherapy, targeted therapy, and/or immunotherapy allowed; for those with BRAF-mutated tumors, progression on BRAF/mitogen-activated protein kinase inhibitor therapy.

Phase 2 (see Figure 15B):

NSCLC-Stage IV NSCLC

- Cohort 1: metastatic NSCLC, PDL1+ve (PD-L1 positivity - tumor proportion score [TPS] ≥50%; PD-L1 IHC 22C3 pharmDx); no prior exposure to PD-1, PD-L1 inhibitors

- Cohort 2: metastatic NSCLC, PDL1-ve (PD-L1 negative - TPS < 1%; PD-L1 IHC 22C3 pharmDx); received chemotherapy and anti-PD-1/PD-L1 therapy in the advanced setting. Prior chemotherapy and PD-1 therapy in the adjuvant setting are allowed, as long as the treatment was completed at least a few months prior to consent.

Melanoma – unresectable stage III or IV cutaneous or unknown primary melanoma

- Melanoma cohort: metastatic disease with prior exposure to anti-PD-1 or anti-PD-L1 agents

Study Duration: How long participants are in the study depends on their treatment. Participants will be followed for overall survival (OS) for 3 years after their last dose of treatment.

Duration of treatment: IMM60 is administered in 3-week cycles for up to several cycles, and pembrolizumab is administered for up to 35 cycles or approximately 2 years.

Duration of follow-up: From the first dose of treatment, or until disease progression or death, whichever comes first, up to 1 year. Participants will be followed for PFS and OS data by their healthcare team during the follow-up period, or until disease progression, further progression, or death, whichever comes first.

End of study: Once the last participant has completed their follow-up period, or until disease progression or death, whichever comes first.

Number of participants:

85-100 evaluable participants will be enrolled in the study (Phase 1: 9 to 18 participants in the initial dose-escalation portion of the study, 12 participants in the following safety portion; Phase 2: 70 participants [45 NSCLC participants with PD-L1>50%, 15 NSCLC participants with PD-L1<1%, 10 PD-1 pretreated melanoma participants]). If any participant in the Phase 1 cohort experiences a dose-limiting toxicity (DLT), an additional 3 participants will be enrolled in that dose level cohort.

Study Groups and Duration:

Issue 1 :

The Phase 1 study will be a standard 3+3 trial design in participants with advanced NSCLC or melanoma:

IMM60 Dose Escalation Safety Cohorting: 3 dose levels of IMM60 will be evaluated (1/3/9 mg/m ² intravenously). If one of the first 3 participants develops a pre-defined DLT, the cohort will be expanded to 6 participants. If another participant in the cohort develops a DLT, the MTD at that dose will be considered exceeded. If more than 1 participant in an expanded cohort is observed to develop a DLT, the MTD will be defined as a dose level lower than that cohort. If more than 1 patient develops a DLT in the lowest IMM60 dose level cohort, TMG will consider de-escalating that dose level. If no participant in the highest dose cohort (9 mg/m ² ) develops a DLT, this cohort will still be expanded to 6 participants.

IMM60 + Pembrolizumab Combination Safety Cohort: Pembrolizumab will be administered in combination with IMM60. The dose of pembrolizumab will be administered as per standard of care (200 mg IV Q3W); the dose of IMM60 (IV) will depend on the IMM60 dose escalation cohort. If no patient experiences a DLT in the 1mg/m ² and 3mg/m ² IMM60 dose escalation cohorts, the IMM60 + Pembrolizumab safety cohort will be opened using the 3mg/m ² IMM60 dosing level and the combined safety cohort will be opened in parallel with the 9mg/m ² IMM60 dose de-escalation cohort. If the combined safety cohort is opened in parallel with the 9mg/m ² IMM60 dose escalation cohort, then if a patient experiences <2 DLTs in the first combined safety cohort in Phase 1, the combined safety cohort will be dose escalated to 9mg/m ² . If any patient in the 1 mg/m ² and 3 mg/m ² IMM60 dose-escalation cohorts develops a DLT, the combined safety cohort will have an IMM60 starting dose level below the IMM60 MTD (referred to as MTD-1) and will be initiated after completion of the 9 mg/m ² dose-escalation cohort, with dose escalation to the cohort where IMM60 is at the MTD.

Dose escalation will be based on toxicity information obtained from evaluable participants in the cohort during the initial 21-day treatment cycle. TMG will review the Phase 1 data, current standard of care, and feasibility to make recommendations regarding the Phase 2 cohort.

Issue 2 :

NSCLC Cohort 1: PD-L1 positive (TPS ≥ 50% PD-L1 IHC 22C3 pharmDx)

NSCLC: Participants will be randomized in a 1:2 ratio to receive pembrolizumab (pembrolizumab alone, 200 mg intravenously Q3W) or a combination of IMM60 + pembrolizumab, 200 mg intravenously Q3W, based on its licensed indication. Based on a review of IMM60 monotherapy activity, NSCLC participants in the pembrolizumab alone cohort who have radiographic progression with clinical worsening or confirmed radiographic progression at two time points at least 4 weeks apart may receive pembrolizumab + IMM60.

Other planned non-randomized phase 2 cohorts include:

NSCLC Cohort 2: PD-L1 negative (TPS < 1% PD-L1 IHC 22C3 pharmDx) NSCLC: Participants will receive one cycle of IMM60 with tumor biopsies before and after to identify any changes in PD-L1 expression. After this cycle, participants will receive the combination of IMM60 + pembrolizumab, 200 mg intravenously, Q3W.

Melanoma Cohort: Melanoma patients who have failed PD-1 inhibitors will receive IMM60 monotherapy at the MTD, administered intravenously Q3W.

Claims (96)

1. A liposome comprising compound A: wherein n is 1 (IMM60), 2 (IMM70) or 3 (IMM80), or a salt, ester, solvate or hydrate thereof, and two or more lipids or salts thereof; Wherein the two or more lipids comprise: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn- Glycerol-3-phospho-rac-glycerol (DSPG); (b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB); (d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC); (e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); or, (f) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL). 2. The liposome of claim 1, wherein n is 1 (IMM60). 3. The liposome of claim 1 or 2, wherein Compound A is present in an amount of about 0.1 wt% to about 20 wt% based on the total weight of the liposome. 4. The liposome of claim 1 or 2, wherein Compound A is present in an amount of about 0.5 wt % to about 15 wt %, or about 1 wt % to about 12 wt %, or about 5 wt % to about 12 wt %, based on the total weight of the liposome. 5. The liposome of any one of claims 1 to 4, wherein the two or more lipids comprise from about 75 wt % to about 99.9 wt %, or from about 80 wt % to about 98 wt %, or from about 80 wt % to about 95 wt %, or from about 85 wt % to about 95 wt %, based on the gross weight of the liposome. 6. The liposome of any one of claims 1 to 5, wherein the mass ratio of Compound A to the two or more lipids is from about 1:5 to about 1:20, or from about 1:7 to about 1:15, or from about 1:7 to about 1:12, or about 1:9. 7. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG). 8. The liposome of claim 7, wherein the DSPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 60 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome. 9. The liposome of claim 7 or 8, wherein the DSPG is present in an amount of about 20 wt % to 60 wt %, or about 30 wt % to about 50 wt %, or about 35 wt % to about 45 wt %, based on the total weight of the liposome. 10. The liposome of any one of claims 7-9, wherein the liposome further comprises cholest-5-en-3β-ol (CHOL). 11. The liposome of claim 10, wherein the CHOL is present in an amount of about 1 wt% to about 20 wt%, or about 5 wt% to about 15 wt%, or about 8 wt% to about 12 wt%, based on the total weight of the liposome. 12. The liposome of claim 10 or 11, wherein the DSPC is present in an amount of about 40% to about 50% by weight, the DSPG is present in an amount of about 30% to about 40% by weight, and the CHOL is present in an amount of about 5% to about 15% by weight. 13. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol). 14. The liposome of claim 13, wherein the POPC is present in an amount of about 40 wt% to 80 wt%, or about 45 wt% to about 65 wt%, or about 50 wt% to about 60 wt%, based on the total weight of the liposome. 15. The liposome of claim 13 or 14, wherein the DC-Chol is present in an amount of about 15 wt % to 55 wt %, or about 20 wt % to about 50 wt %, or about 25 wt % to about 35 wt %, based on the total weight of the liposome. 16. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecylammonium bromide (DDAB). 17. The liposome of claim 16, wherein the POPC is present in an amount of about 40 wt% to 80 wt%, or about 45 wt% to about 65 wt%, or about 50 wt% to about 60 wt%, based on the total weight of the liposome. 18. The liposome of claim 1 or 17, wherein the DC-Chol is present in an amount of about 15 wt % to 55 wt %, or about 20 wt % to about 50 wt %, or about 30 wt % to about 40 wt %, based on the total weight of the liposome. 19. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC). 20. The liposome of claim 19, wherein the EPG is present in an amount of about 40 wt% to 80 wt%, or about 55 wt% to about 75 wt%, or about 60 wt% to about 70 wt%, based on the total weight of the liposome. 21. The liposome of claim 19 or 20, wherein the EPC is present in an amount of about 10 wt% to 50 wt%, or about 15 wt% to about 40 wt%, or about 15 wt% to about 30 wt%, based on the total weight of the liposome. 22. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). 23. The liposome of claim 22, wherein the POPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 65 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome. 24. The liposome of claim 22 or 23, wherein the DOTAP is present in an amount of about 20 wt% to 50 wt%, or about 25 wt% to about 45 wt%, or about 35 wt% to about 45 wt%, based on the total weight of the liposome. 25. The liposome of any one of claims 1 to 6, wherein the two or more lipids comprise 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL). 26. liposomes as claimed in claim 25, wherein the DMPG is present in an amount of about 40 wt % to 80 wt %, or about 50 wt % to about 75 wt %, or about 60 wt % to about 70 wt %, based on the total weight of the liposome. 27. The liposome of claim 25 or 26, wherein the CHOL is present in an amount of about 10 wt% to 50 wt%, or about 15 wt% to about 35 wt%, or about 15 wt% to about 30 wt%, based on the total weight of the liposome. 28. The liposome of any one of claims 1 to 27, wherein the liposome further comprises a liposome buffer. 29. The liposome of claim 28, wherein the pH of the liposome buffer is about 7, or about 6.3 to about 6.7. 30. The liposome of claim 29, wherein the liposome buffer comprises NaCl and sodium phosphate. 31. The liposome of any one of claims 28-30, wherein the liposome buffer further comprises sucrose. 32. The liposome of any one of claims 1 to 31, wherein the liposome further comprises at least one antigen. 33. The liposome of claim 32, wherein the at least one antigen comprises a member selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, tumor antigens, and mixtures thereof. 34. The liposome of claim 32 or 33, wherein the at least one antigen comprises at least one tumor antigen. 35. The liposome of claim 34, wherein the tumor antigen is selected from the group consisting of: (a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, (b) an antigenic fragment of any one of (a), and (c) A mixture of any one of (a) and/or (b). 36. The liposome of any one of claims 31-35, wherein the antigen is present in the liposome in an amount of about 1 g antigen to 1-20 g Compound A. 37. The liposome of any one of claims 1 to 36, further comprising at least one therapeutic agent. 38. A composition comprising the liposome of any one of claims 1-37 and a pharmaceutically acceptable diluent, excipient, adjuvant or carrier. 39. The composition of claim 38, wherein the average diameter of the liposomes is less than 200 nm. 40. The composition of claim 38, wherein the average diameter of the liposomes is in the range of about 50 nm to 200 nm, or about 50 nm to about 170 nm, or about 75 nm to about 145 nm, or about 90 nm to about 130 nm. 41. The composition of claim 38, wherein the average diameter of the liposome is 110 nm ± 60 nm, 110nm±40nm or 110nm±20nm. 42. The composition of any one of claims 38 to 41, wherein the liposomes have a polydispersity index (PdI) of less than or equal to about 0.40, or less than or equal to about 0.35, or less than or equal to about 0.30, or less than or equal to about 0.25, or less than or equal to about 0.20, or less than or equal to about 0.15. 43. The composition of any one of claims 38 to 42, wherein the composition further comprises at least one antigen. 44. The composition of claim 43, wherein the at least one antigen comprises a member selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, tumor antigens, and mixtures thereof. 45. The composition of claim 43 or 44, wherein the at least one antigen comprises at least one tumor antigen. 46. The composition of claim 45, wherein the tumor antigen is selected from the group consisting of: (a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, (b) an antigenic fragment of any one of (a), and (c) A mixture of any one of (a) and/or (b). 47. The composition of claim 45, wherein the tumor antigen comprises NY-ESO-1 or an antigenic fragment thereof. 48. The composition of any one of claims 38 to 47, further comprising at least one therapeutic agent. 49. The composition of claim 48, wherein the at least one therapeutic agent is selected from the group consisting of an immunomodulator, a Toll-like receptor agonist, a Nod ligand, an antiviral agent, an antifungal agent, an antibiotic, an antiviral antibody, a cancer immunotherapeutic agent, a chemotherapeutic agent, a kinase inhibitor, a cytotoxic agent, an anti-asthmatic agent, an antihistamine, an anti-inflammatory agent, a vaccine adjuvant, a second liposome, an artificial antigen presenting cell, a cytokine or chemokine blocking antibody, and combinations thereof. 50. A method of stimulating an immune response in a mammalian subject, the method comprising administering to the subject the composition of any one of claims 38 to 49. 51. The method of claim 50, further comprising the step of heating said composition prior to said administering step for a time and at a temperature sufficient to reduce the average particle size or PdI of said liposome composition. 52. The method of claim 50, further comprising the step of heating the composition at a temperature of about 55°C to about 65°C for 5-15 minutes prior to the applying step. 53. A composition as described in any one of claims 38 to 49 for stimulating an immune response in a mammalian subject. 54. Use of a composition as described in any one of claims 38 to 49 for stimulating an immune response in a mammalian subject. 55. A liposome as claimed in any one of claims 1 to 37 or a composition as claimed in any one of claims 38 to 49 for use in the preparation of a medicament for stimulating an immune response in a mammalian subject. 56. A composition or use as described in any one of claims 53 to 55, wherein before the composition is used to stimulate the immune response, it is subjected to a heating step. 57. The method or use of any one of claims 50-56, wherein the mammalian subject is a human. 58. The method or use of any one of claims 50 to 57, wherein the mammalian subject has cancer. 59. A method of treating a mammalian subject having cancer, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 38 to 49. 60. The method or use of claim 58 or 59, wherein the composition is administered by subcutaneous, intravenous or intratumoral injection. 61. The method of claim 50 or 60, further comprising the step of heating said composition prior to said administering step for a time and at a temperature sufficient to reduce the average particle size or PdI of said liposome composition. 62. The liposome of any one of claims 1 to 37 or the composition of any one of claims 38 to 49 for use in treating cancer in a mammalian subject. 63. A liposome as described in any one of claims 1 to 37 or a composition as described in any one of claims 38 to 49 for use in the preparation of a medicament for treating cancer in a mammalian subject. 64. A composition or use as described in any one of claims 61 to 62, wherein before the composition is used to stimulate the immune response, it is subjected to a heating step. 65. The method or use of any one of claims 58 to 64, wherein the cancer is selected from the group consisting of basal cell carcinoma, breast cancer, leukemia, Burkitt's lymphoma, colon cancer, esophageal cancer, bladder cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hairy cell leukemia, Wilms' tumor, thyroid cancer, thymoma and thymic carcinoma, testicular cancer, T-cell lymphoma, prostate cancer, non-small cell lung cancer, liver cancer, renal cell carcinoma, melanoma, and combinations thereof. 66. The method or use of any one of claims 58 to 64, wherein the cancer comprises melanoma. 67. The method or use of any one of claims 58 to 64, wherein the cancer comprises non-small cell lung cancer. 68. The method or use of any one of claims 58 to 67, further comprising administering to the subject at least one further treatment or therapy selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent and radiation therapy. 69. The method or use of any one of claims 58-68, further comprising administering or using at least one checkpoint inhibitor to the subject. 70. The method or use of claim 69, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. 71. The method or use of claim 70, wherein the immune checkpoint inhibitor comprises at least one PD-1 inhibitor selected from Pembrolizumab, Nivolumab, Cemiplimab, and Spartalizumab. 72. The method or use of claim 70, wherein the immune checkpoint inhibitor comprises at least one PD-L1 inhibitor selected from Atezolizumab, Avelumab, and Durvalumab. 73. The method or use of claim 70, wherein the immune checkpoint inhibitor comprises at least one CTLA-4 inhibitor selected from Ipilimumab and Tremelimumab. 74. The method or use of any one of claims 68-73, wherein the composition and the further treatment or therapy are administered simultaneously. 75. The method or use of any one of claims 68-73, wherein the composition and the further treatment or therapy are administered separately. 76. A method for preparing liposomes, wherein the liposomes contain compound A wherein n is 1 (IMM60), 2 (IMM70) or 3 (IMM80), or a salt, ester, solvate or hydrate thereof, and two or more lipids or salts thereof; the method comprising: (A) mixing compound A and two or more lipids to form multilamellar vesicles, Wherein the two or more lipids comprise: (a) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG); (b) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); (c) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and dimethyldioctadecyl ammonium bromide (DDAB); (d) L-α-phosphatidylglycerol (EPG) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC); (e) 2-oleoyl-1-palmitoylglycero-3-phosphocholine (POPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); or (f) 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cholest-5-en-3β-ol (CHOL), and (B) Multilamellar vesicles are extruded through the membrane to form liposomes. 77. The method of claim 76, further comprising (c) purifying the liposomes. 78. The method of claim 77, wherein said purifying comprises filtration. 79. The method of claim 77, wherein the purification comprises ultrafiltration, centrifugation, dialysis, diafiltration, or tangential flow filtration. 80. The method of claim 78, comprising filtering with a filter having a pore size of 400 nm, or less than about 350 nm, or less than about 300 nm, or less than about 250 nm, or less than about 220 nm, or less than about 200 nm, or less than about 150 nm. 81. The method of claim 78, comprising filtering with a filter having a pore size of about 50 nm to about 250 nm, or about 50 nm to about 150 nm, or about 60 nm to about 100 nm, or about 80 nm. 82. The method of claim 78, comprising filtering with a filter having a molecular weight cutoff (MWCO) of 1000 kD, or 800 kD, or 600 kD, or 500 kD, or 400 kD, or 300 kD, or 250 kD. 83. The method of any one of claims 7 to 82, wherein n is 1 (IMM60). 84. The method of any one of claims 7-83, wherein Compound A is present in an amount of about 0.1 wt % to about 20 wt %, or about 0.5 wt % to about 15 wt %, or about 1 wt % to about 12 wt %, or about 5 wt % to about 12 wt %, based on the total weight of the liposome. 85. methods as described in any one of claims 7-84, wherein based on the gross weight of described liposome, described two or more lipids comprise about 75 wt % to about 99.9 wt %, or about 80 wt % to about 98 wt %, or about 80 wt % to about 95 wt %, or about 85 wt % to about 95 wt %. 86. methods as described in any one of claims 7-81, wherein the mass ratio of Compound A to the two or more lipids is in the range of about 1:5 to about 1:20, or about 1:7 to about 1:15, or about 1:7 to about 1:12, or about 1:9. 87. The method of any one of claims 7-86, wherein the two or more lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG). 88. The method of claim 87, wherein the DSPC is present in an amount of about 40 wt % to 80 wt %, or about 40 wt % to about 60 wt %, or about 45 wt % to about 55 wt %, based on the total weight of the liposome. 89. The method of claim 87 or 88, wherein the DSPG is present in an amount of about 20 wt% to 60 wt%, or about 30 wt% to about 50 wt%, or about 35 wt% to about 45 wt%, based on the total weight of the liposome. 90. The method of any one of claims 7-84, wherein the two or more lipids further comprise CHOL. 91. The method of any one of claims 7-90, wherein step (A) further comprises mixing at least one antigen with the compound A and the lipid. 92. The method of claim 91, wherein the at least one antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, and/or a tumor antigen. 93. The method of claim 91 or 92, wherein the at least one antigen comprises at least one tumor antigen. 94. The liposome of claim 93, wherein the tumor antigen is selected from the group consisting of: (a)P1A, MUC1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, CAGE, LB33/MUM-1, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), brain glycogen phosphorylase, MAGE-C1/CT7, MAGE-C2, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-i, NY-ESO-1, PRAME, PSMA, tyrosinase, melan-A, XAGE, (b) an antigenic fragment of any one of (a), and (c) A mixture of any one of (a) and/or (b). 95. The method of any one of claims 7-94, wherein step (A) further comprises mixing at least one therapeutic agent with the lipid and the Compound A. 96. A liposome prepared by the method of any one of claims 76-95.