阅读说明：本技术 治疗癌症的方法 (Methods of treating cancer ) 是由 C·哈维 D·劳 A·E·汉森 M·Y·范 于 2019-05-14 设计创作，主要内容包括：本发明提供了治疗癌症的方法和用于选择癌症治疗途径的方法。(The present invention provides methods of treating cancer and methods for selecting a therapeutic pathway for cancer.)

1. One or more anti-cancer therapies or an anti-ICOS agonist for use in a method of treating cancer in a subject in need thereof, the method comprising (i) administering one or more doses of the one or more anti-cancer therapies to the subject, (ii) obtaining one or more peripheral blood test samples from the subject after the administration, (iii) measuring the ICOS and/or T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, (iv) determining whether a CD4+ T cell population having elevated ICOS and/or T-beta levels is present in any of the one or more peripheral blood test samples when compared to a control, and (v) if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having elevated ICOS and/or T-beta levels, administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) the anti-ICOS agonist.

2. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 1, wherein step (iii) comprises measuring the ICOS level of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having an elevated ICOS level is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS level.

3. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 1 or 2, wherein step (iii) comprises measuring the T-beta level of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population with an elevated T-beta level is present in any one of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any one of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population with an elevated T-beta level.

4. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 1, wherein step (iii) comprises measuring ICOS and T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having elevated ICOS and T-beta levels is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having elevated ICOS and/or T-beta levels.

5. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 1, wherein step (iii) comprises measuring ICOS and T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having elevated ICOS and T-beta levels is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having elevated ICOS and T-beta levels.

6. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 5, wherein said one or more anti-cancer therapies comprise immunotherapy.

7. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 6, wherein said one or more anti-cancer therapies comprise an anti-CTLA-4 antagonist antibody.

8. The one or more anti-cancer therapies or anti-ICOS agonists for use according to claim 7, wherein the anti-CTLA-4 antagonist antibody is selected from the group consisting of: ipilimumab, tremelimumab and BMS-986249.

9. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 8, wherein the anti-CTLA-4 antagonist antibody is ipilimumab.

10. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 9, wherein said one or more anti-cancer therapies comprise an anti-PD-1 or anti-PD-L1 antagonist antibody.

11. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 10, wherein the anti-PD-1 or anti-PD-L1 antagonist antibody is selected from the group consisting of: abameluzumab, alemtuzumab, CX-072, pembrolizumab, nivolumab, cimirapril mab, sibadazumab, tiramerizumab, JNJ-63723283, Jennuzumab, AMP-514, AGEN2034, Dewaruzumab and JNC-1.

12. One or more anti-cancer therapies or anti-ICOS agonists for use according to claim 11, wherein said anti-PD-1 or anti-PD-L1 antagonist antibody is selected from the group consisting of: pembrolizumab, nivolumab, alemtuzumab, avizumab, and Devolumab.

13. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 12, wherein said one or more anti-cancer therapies or said anti-ICOS agonist comprises an anti-ICOS agonist antibody.

14. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 13, wherein said anti-ICOS agonist antibody is selected from the group consisting of: JTX-2011, BMS-986226, and GSK 3359609.

15. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 14, wherein said one or more anti-cancer therapies comprise one or more of the therapies listed in table 2.

16. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 15, wherein said one or more anti-cancer therapies comprise chemotherapy.

17. One or more anti-cancer therapies or anti-ICOS agonists for use according to claim 16, wherein said chemotherapy is selected from the group consisting of: capecitabine, cyclophosphamide, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine, irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, nabumeclitol, pemetrexed, vinorelbine, vincristine, erlotinib, afatinib, gefitinib, crizotinib, darafinib, tremetinib, vemurafenib, and cobicistinib.

18. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 17, wherein said one or more anti-cancer therapies comprises radiation therapy.

19. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 18, wherein step (v) comprises administering to the subject an anti-ICOS antibody agonist if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS and/or T-beta level.

20. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 13 to 19, wherein the anti-ICOS antibody agonist comprises at least one CDR selected from the group consisting of: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:10, wherein one or more of the CDRs comprise 1 or 2 amino acid substitutions.

21. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 20, wherein the anti-ICOS antibody agonist comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

22. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 13 to 21, wherein said anti-ICOS antibody agonist comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID No. 1, and/or (b) a light chain comprising the amino acid sequence of SEQ ID No. 2.

23. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 22, wherein said one or more anti-cancer therapies comprise any combination of at least two of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

24. One or more anti-cancer therapies or anti-ICOS agonists for use according to any one of claims 1 to 23, wherein said one or more anti-cancer therapies comprise any combination of at least three of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

25. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 24, wherein the one or more anti-cancer therapies are administered two or more times prior to obtaining the one or more peripheral blood test samples.

26. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 25, wherein the one or more anti-cancer therapies are administered three or more times prior to obtaining the one or more peripheral blood test samples.

27. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 26, wherein the one or more anti-cancer therapies are administered four or more times prior to obtaining the one or more peripheral blood test samples.

28. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 27, wherein the one or more anti-cancer therapies are administered five or more times prior to obtaining the one or more peripheral blood test samples.

29. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 28, wherein the obtaining of the one or more peripheral blood test samples is performed less than 4 weeks after the one or more administrations of the dose of the one or more anti-cancer therapies.

30. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 29, wherein the obtaining of the one or more peripheral blood test samples is performed less than 3 weeks after the one or more administrations of the dose of the one or more anti-cancer therapies.

31. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 30, wherein the obtaining of the one or more peripheral blood test samples is performed less than 2 weeks after the one or more administrations of the dose of the one or more anti-cancer therapies.

32. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 31, wherein the obtaining of the one or more peripheral blood test samples is performed less than one week after the one or more administrations of the dose of the one or more anti-cancer therapies.

33. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 32, wherein a dose of the one or more anti-cancer therapies is administered multiple times at regular intervals.

34. One or more anti-cancer therapies or anti-ICOS agonists for use according to claim 33, wherein said regular intervals are selected from the group consisting of: weekly, biweekly, every three weeks, every four weeks, every six weeks, every nine weeks, and every twelve weeks.

35. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 33 or 34, wherein the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein a test sample is obtained concurrently with one or more of the administrations.

36. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 33 or 34, wherein the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein test samples are obtained during the time between the plurality of administrations.

37. The one or more anti-cancer therapies or anti-ICOS agonist for use of any one of claims 33-36, wherein the method further comprises discontinuing administration of the one or more anti-cancer therapies if a CD4+ T cell population having an elevated ICOS and/or T-beta level compared to a control is not detected in any of the peripheral blood test samples after administration of the one or more anti-cancer therapies for four or more intervals.

38. The one or more anti-cancer therapies or anti-ICOS agonist for use of claim 37, wherein the method further comprises discontinuing administration of the one or more anti-cancer therapies if a peripheral blood test sample is obtained after administration of the one or more anti-cancer therapies for five or more, six or more, seven or more, eight or more, nine or more, or ten or more intervals, based on which it is determined that there is no population of CD4+ T cells having elevated ICOS and/or T-beta levels compared to a control.

39. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 38, wherein said method further comprises storing a portion of one or more of said peripheral blood test samples.

40. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 39, wherein a portion of said CD4+ T cells having elevated ICOS and/or T-beta levels are isolated from one or more of said peripheral blood test samples and stored under conditions suitable for maintaining the viability of said CD4+ T cells.

41. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 40, wherein the stored CD4+ T cells are stored in a cell culture medium.

42. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 40 or 41, wherein the stored CD4+ T cells are stored at a concentration greater than 100,000 cells/mL.

43. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 42, wherein the stored CD4+ T cells are stored at a concentration of between 100,000 cells/mL and 1 hundred million cells/mL.

44. A suspension of CD4+ T cells obtained according to the use or method of any one of claims 40 to 43.

45. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 43, wherein the control comprises a peripheral blood test sample, optionally obtained from the subject prior to one or more administrations of the one or more anti-cancer therapies to the subject.

46. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 43, wherein said control comprises a peripheral blood sample obtained from a healthy individual who has not received said one or more anti-cancer therapies.

47. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 43, 45 or 46, wherein determining ICOS and/or T-beta levels comprises the use of an immunoassay.

48. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 47, wherein said immunoassay comprises the detection of ICOS using an antibody that binds to the intracellular domain of ICOS.

49. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 48, wherein said antibody comprises the heavy chain variable region sequence of SEQ ID NO:27 and the light chain variable region sequence of SEQ ID NO: 31; or the antibody comprises the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

50. The one or more anti-cancer therapies or anti-ICOS agonist for use according to claim 48, wherein said antibody cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO:27 and the light chain variable region sequence of SEQ ID NO: 31; or cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

51. The one or more anti-cancer therapies or anti-ICOS agonist for use of any one of claims 1-43 or 45-50, further comprising measuring ICOS and/or T-beta levels of CD8+ T cells present in the one or more peripheral blood test samples, wherein a population of CD8+ T cells having elevated ICOS and/or T-beta levels relative to a control is not detected in the sample.

52. One or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 43 or 45 to 51, wherein the cancer is selected from gastric cancer, breast cancer optionally Triple Negative Breast Cancer (TNBC), non-small cell lung cancer (NSCLC), melanoma, Renal Cell Carcinoma (RCC), bladder cancer, endometrial cancer, diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, ovarian cancer and Head and Neck Squamous Cell Carcinoma (HNSCC).

53. The one or more anti-cancer therapies or anti-ICOS agonist for use according to any one of claims 1 to 43 or 45 to 52, wherein the subject is a human patient.

54. The one or more anti-cancer therapies or anti-ICOS agonist for use of any one of claims 1 to 43 or 45 to 53, wherein said population of CD4+ T cells having elevated ICOS and/or T-beta levels comprises a novel population of CD4+ T cells alone induced by said one or more anti-cancer therapies.

55. A method of producing an expanded population of CD4+ T cells with elevated ICOS expression, the method comprising culturing the CD4+ T cell suspension of claim 44 under initial culture conditions suitable for expanding the CD4+ T cell population.

56. The method of claim 55, wherein the initial conditions suitable for expansion of the population of CD4+ T cells comprise contacting the CD4+ T cell suspension with a CD3 agonist.

57. The method of claim 56, wherein the CD3 agonist is an anti-CD 3 antibody (e.g., OKT 3).

58. The method of any one of claims 55 to 57, wherein the initial conditions suitable for expansion of the CD4+ T cell population comprise contacting the suspension with one or more of an anti-PD-1 antibody antagonist, an anti-CTLA-4 antibody and an ICOS agonist.

59. The method of any one of claims 55 to 58, wherein the initial conditions suitable for expansion of the CD4+ T cell population comprise contacting the suspension with one or more compounds (e.g., two or more or all three) selected from the group consisting of IL-2, IL-12, and anti-IL-4.

60. The method of any one of claims 55 to 58, wherein the initial conditions suitable for expansion of the CD4+ T cell population comprise contacting the suspension with an anti-CD 28 antibody agonist.

61. The method of any one of claims 57-60, wherein the CD3 agonist and anti-CD 28 agonist are present as a tetrameric antibody complex.

62. The method of any one of claims 55 to 61, wherein the CD4+ T cell suspension is incubated under the initial culture conditions for a period of time between one and five days (e.g., about 1 day, 2 days, 3 days, 4 days, or five days).

63. The method of claim 62, further comprising incubating the CD4+ T cell suspension under second culture conditions suitable for expansion of the CD4+ T cell population.

64. The method of claim 63, wherein the cells are washed prior to applying the second culture conditions.

65. The method of claim 63 or 64, wherein the second culture conditions comprise contacting the cell suspension with one or more of an anti-PD-1 antibody antagonist, an anti-CTLA-4 antibody, and an ICOS agonist.

66. The method of any one of claims 63-65, wherein the second culture conditions comprise contacting the cell suspension with one or more compounds (e.g., two or more or all three) selected from the group consisting of IL-2, IL-12, and anti-IL-4.

67. The method of any one of claims 63-66, wherein the second culture conditions comprise contacting the cell suspension with an anti-CD 28 antibody agonist.

68. The method of any one of claims 63-66, wherein the second culture conditions do not comprise contacting the cell suspension with a CD3 agonist and/or a CD28 agonist.

69. The method of any one of claims 63-68, wherein the second culture condition is maintained for between 1 day and 5 days (e.g., for 1 day, 2 days, 3 days, 4 days, or 5 days).

70. A cell suspension produced by the method of any one of claims 55-69.

71. The cell suspension of claim 70, wherein the T cell suspension of claim 44 is optionally isolated from the subject for use in a method of treating cancer in a subject in need thereof, the method comprising administering the cell suspension to the subject.

72. A method of treating cancer in a subject in need thereof, the method comprising (i) administering one or more doses of one or more anti-cancer therapies to the subject, (ii) obtaining one or more peripheral blood test samples from the subject after the administering, (iii) measuring ICOS and/or T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, (iv) determining whether a CD4+ T cell population having an elevated ICOS and/or T-beta level is present in any of the one or more peripheral blood test samples when compared to a control, and (v) administering (a) one or more additional doses of the one or more anti-cancer therapies to the subject if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS and/or T-beta level, or (b) an anti-ICOS agonist.

73. A method for determining whether a subject may benefit from (a) continued treatment with one or more anti-cancer therapies or (b) treatment with an anti-ICOS agonist, the method comprising determining ICOS and/or T-beta levels of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of increased ICOS and/or T-beta levels relative to a control indicates that the subject may benefit from the continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

74. The method of claim 73, wherein the method comprises determining the ICOS level of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of an increased ICOS level relative to a control indicates that the subject may benefit from the continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

75. The method of claim 73 or 74, wherein the method comprises determining T-beta levels of peripheral CD4+ T cells in a blood sample of the subject, wherein detection of increased T-beta levels relative to a control indicates that the subject may benefit from the continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

76. The method of claim 73, wherein the method comprises determining ICOS and T-beta levels of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of increased ICOS or T-beta levels relative to a control indicates that the subject may benefit from the continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

77. The method of any one of claims 73-76, wherein the one or more anti-cancer therapies comprise immunotherapy.

78. The method of any one of claims 73-77, wherein the one or more anti-cancer therapies comprise anti-CTLA-4 antagonist antibodies.

79. The method of claim 78, wherein the anti-CTLA-4 antagonist antibody is selected from the group consisting of: ipilimumab, tremelimumab and BMS-986249.

80. The method of claim 79, wherein the anti-CTLA-4 antagonist antibody is ipilimumab.

81. The method of any one of claims 73-80, wherein the one or more anti-cancer therapies comprise an anti-PD-1 or anti-PD-L1 antagonist antibody.

82. The method of claim 81, wherein the anti-PD-1 or anti-PD-L1 antagonist antibody is selected from the group consisting of: abameluzumab, alemtuzumab, CX-072, pembrolizumab, nivolumab, cimirapril mab, sibadazumab, tiramerizumab, JNJ-63723283, Jennuzumab, AMP-514, AGEN2034, Dewaruzumab and JNC-1.

83. The method of claim 82, wherein the anti-PD-1 or anti-PD-L1 antagonist antibody is selected from the group consisting of: pembrolizumab, nivolumab, alemtuzumab, avizumab, and Devolumab.

84. The method of any one of claims 73-83, wherein the one or more anti-cancer therapies or the anti-ICOS agonist comprises an anti-ICOS agonist antibody.

85. The method of claim 84, wherein the anti-ICOS agonist antibody is selected from the group consisting of: JTX-2011, BMS-986226, and GSK 3359609.

86. The method of any one of claims 73-85, wherein said one or more anti-cancer therapies comprise one or more therapies listed in Table 2.

87. The method of any one of claims 73-86, wherein the one or more anti-cancer therapies comprise chemotherapy.

88. The method of claim 87, wherein the chemotherapy is selected from the group consisting of: capecitabine, cyclophosphamide, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine, irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, nabumeclitol, pemetrexed, vinorelbine, vincristine, erlotinib, afatinib, gefitinib, crizotinib, darafinib, tremetinib, vemurafenib, and cobicistinib.

89. The method of any one of claims 73-88, wherein the one or more anti-cancer therapies comprise radiation therapy.

90. The method of any one of claims 73-89, wherein step (v) comprises administering to the subject an anti-ICOS antibody agonist if any of the one or more peripheral blood test samples is determined to comprise a population of CD4+ T cells having elevated ICOS and/or T-beta levels.

91. The method of any one of claims 73-90, wherein the anti-ICOS antibody agonist comprises at least one CDR selected from the group consisting of: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:10, wherein one or more of the CDRs comprise 1 or 2 amino acid substitutions.

92. The method of claim 91, wherein the anti-ICOS antibody agonist comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

93. The method of any one of claims 73-92, wherein the anti-ICOS antibody agonist comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO 1, and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO 2.

94. The method of any one of claims 73-93, wherein the one or more anti-cancer therapies comprise any combination of at least two of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

95. The method of any one of claims 73-94, wherein the one or more anti-cancer therapies comprise any combination of at least three of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

96. The method of any one of claims 73-95, wherein said one or more anti-cancer therapies are administered two or more times prior to obtaining said one or more peripheral blood test samples.

97. The method of claim 96, wherein the one or more anti-cancer therapies are administered three or more times prior to obtaining the one or more peripheral blood test samples.

98. The method of claim 97, wherein the one or more anti-cancer therapies are administered four or more times before the one or more peripheral blood test samples are obtained.

99. The method of claim 98, wherein the one or more anti-cancer therapies are administered five or more times prior to obtaining the one or more peripheral blood test samples.

100. The method of any one of claims 73-99, wherein obtaining said one or more peripheral blood test samples is performed less than 4 weeks after said administering one or more doses of said one or more anti-cancer therapies.

101. The method of claim 100, wherein obtaining said one or more peripheral blood test samples is performed less than 3 weeks after said one or more doses of said one or more anti-cancer therapies.

102. The method of claim 101, wherein obtaining said one or more peripheral blood test samples is performed less than 2 weeks after said one or more doses of said one or more anti-cancer therapies.

103. The method of claim 102, wherein obtaining said one or more peripheral blood test samples is performed less than one week after said one or more doses of said one or more anti-cancer therapies.

104. The method of any one of claims 73-103, wherein a dose of the one or more anti-cancer therapies is administered multiple times at regular intervals.

105. The method of claim 104, wherein the regular intervals are selected from the group consisting of: weekly, biweekly, every three weeks, every four weeks, every six weeks, every nine weeks, and every twelve weeks.

106. The method of claim 104 or 105, wherein the obtaining of one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein a test sample is obtained concurrently with one or more of the administering.

107. The method of claim 104 or 105, wherein the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein test samples are obtained during the time between the plurality of administrations.

108. The method of any one of claims 104-107, wherein the method further comprises discontinuing administration of the one or more anti-cancer therapies if a population of CD4+ T cells having an elevated ICOS and/or T-beta level compared to a control is not detected in any of the peripheral blood test samples after administration of the one or more anti-cancer therapies for four or more intervals.

109. The method of claim 108, wherein the method further comprises discontinuing administration of the one or more anti-cancer therapies if a peripheral blood test sample is obtained after administration of the one or more anti-cancer therapies for five or more, six or more, seven or more, eight or more, nine or more, or ten or more intervals, based on which it is determined that there is no population of CD4+ T cells having elevated ICOS and/or T-beta levels compared to a control.

110. The method of any one of claims 73-109, wherein the method further comprises storing a portion of one or more of the peripheral blood test samples.

111. The method of any one of claims 73-110, wherein a portion of the CD4+ T cells having elevated ICOS and/or T-beta levels are isolated from one or more of the peripheral blood test samples and stored under conditions suitable for maintaining viability of the CD4+ T cells.

112. The method of claim 111, wherein the stored CD4+ T cells are stored in cell culture medium.

113. The method of claim 111 or 112, wherein the stored CD4+ T cells are stored at a concentration greater than 100,000 cells/mL.

114. The method of claim 113, wherein the stored CD4+ T cells are stored at a concentration of between 100,000 cells/mL and 1 hundred million cells/mL.

115. A CD4+ T cell suspension obtained according to the method of any one of claims 111-114.

116. The method of any one of claims 73 to 114, wherein the control comprises a peripheral blood test sample, optionally obtained from the subject prior to one or more administrations of the one or more anti-cancer therapies to the subject.

117. The method of any one of claims 73-114, wherein the control comprises a peripheral blood sample obtained from a healthy individual who has not received the one or more anti-cancer therapies.

118. The method of any one of claims 73-114, 116, or 117, wherein determining ICOS and/or T-beta levels comprises using an immunoassay.

119. The method of claim 118, wherein the immunoassay comprises detection of ICOS using an antibody that binds to the intracellular domain of ICOS.

120. The method of claim 119, wherein the antibody comprises the heavy chain variable region sequence of SEQ ID No. 27 and the light chain variable region sequence of SEQ ID No. 31; or the antibody comprises the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

121. The method of claim 119, wherein the antibody cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID No. 27 and the light chain variable region sequence of SEQ ID No. 31; or cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

122. The method of any one of claims 73-114 or 116-121, further comprising measuring ICOS and/or T-beta levels of CD8+ T cells present in the one or more peripheral blood test samples, wherein no CD8+ T cell population having elevated ICOS and/or T-beta levels relative to a control is detected in the sample.

123. The method of any one of claims 73 to 114 or 116 to 122, wherein the cancer is selected from gastric cancer, breast cancer optionally Triple Negative Breast Cancer (TNBC), non-small cell lung cancer (NSCLC), melanoma, Renal Cell Carcinoma (RCC), bladder cancer, endometrial cancer, diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, ovarian cancer and Head and Neck Squamous Cell Carcinoma (HNSCC).

124. The method of any one of claims 73-114 or 116-123, wherein the subject is a human patient.

125. The method of any one of claims 73-114 or 116-124, wherein the population of CD4+ T cells with elevated ICOS and/or T-beta levels comprises a novel population of CD4+ T cells alone induced by the one or more anti-cancer therapies.

Technical Field

The present invention relates to methods of treating cancer and methods of selecting a therapeutic pathway for cancer.

Background

ICOS (inducible T cell co-stimulatory factor; CD278) is a member of the B7/CD28/CTLA-4 immunoglobulin superfamily, with the tag being specifically expressed on T cells. Unlike CD28, which is constitutively expressed on T cells and provides the costimulatory signals required to fully activate resting T cells, ICOS is only expressed following initial T cell activation.

ICOS has been implicated in different aspects of T cell responses (reviewed in Simpson et al, curr. opin. immunol.22:326-332, 2010). It plays a role in germinal center formation, T/B cell cooperation and immunoglobulin class switching. ICOS-deficient mice show impaired germinal center formation and have reduced interleukin IL-10 production. These defects have been clearly associated with defects in T follicle helper cells. ICOS also plays a role in the development and function of other T cell subsets, including Th1, Th2, and Th 17. Notably, ICOS co-stimulated T cell proliferation and cytokine secretion associated with both Th1 and Th2 cells. Thus, ICOS knockout mice display impaired development of autoimmune phenotypes in a variety of disease models, including diabetes (Th1), airway inflammation (Th2), and EAE neuroinflammation model (Th 17).

In addition to its role in regulating T effector (Teff) cell function, ICOS also regulates T regulatory cells (tregs). ICOS is expressed at high levels on tregs and is associated with the homeostasis and function of tregs.

Upon activation, ICOS (disulfide-linked homodimer) induces signaling through PI3K and the AKT pathway. Subsequent signaling events result in the expression of lineage specific transcription factors (e.g., T-beta, GATA-3) and, in turn, have an effect on T cell proliferation and survival.

ICOS ligand (ICOSL; B7-H2; B7RP 1; CD 275; GL50), also a member of the B7 superfamily, is the sole ligand for ICOS and is expressed on the cell surface of B cells, macrophages and dendritic cells. ICOSL functions as a non-covalently linked homodimer on the cell surface when it interacts with ICOS. Human ICOSL, although not mouse ICOSL, has been reported to bind to human CD28 and CTLA-4 (Yao et al, Immunity 34: 729-.

T-beta (T-box expressed in T-cells) is a member of the T-box family of transcription factors and is a lineage-defined transcription factor that is selectively expressed in thymocytes and Th1 cells. It initiates development of the Th1 lineage from naive Th precursor cells both by activating the Th1 genetic program and by suppressing the opposite Th2 and Th17 genetic programs. T-beta activates transcription of a set of genes important for Th1 cell function, including those encoding interferon gamma (IFN- γ) and chemokine receptor CXCR3, and can also redirect polarized Th2 cells into the Th1 pathway. T-beta also controls IFN-gamma production in CD8+ T cells as well as in cells of the innate immune system (e.g., NK cells and dendritic cells). Expression of human T-beta correlates with IFN-gamma expression in Th1 and natural killer cells, suggesting a role for this gene in initiating development of the Th1 lineage from naive Th precursor cells (Szabo et al, Cell 100(6): 665-.

Disclosure of Invention

The invention provides one or more anti-cancer therapies or anti-ICOS agonists for use in a method of treating cancer in a subject in need thereof, the method comprising (i) administering one or more doses of one or more anti-cancer therapies to the subject, (ii) obtaining one or more peripheral blood test samples from the subject following administration, (iii) measuring the ICOS and/or T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, (iv) determining whether a CD4+ T cell population having an elevated ICOS and/or T-beta level is present in any of the one or more peripheral blood test samples when compared to a control, and (v) if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS and/or T-beta level, administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) the anti-ICOS agonist.

The invention also provides a method of treating cancer in a subject in need thereof (e.g., a human patient), the method comprising (i) administering one or more doses of one or more anti-cancer therapies to the subject, (ii) obtaining one or more peripheral blood test samples from the subject after administration, (iii) measuring the ICOS and/or T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, (iv) determining whether a CD4+ T cell population having an elevated ICOS and/or T-beta level is present in any one of the one or more peripheral blood test samples when compared to a control, and (v) if any one of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS and/or T-beta level, administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist.

In some embodiments, step (iii) comprises measuring the ICOS level of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having an elevated ICOS level is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS level.

In some embodiments, step (iii) comprises measuring the T-beta level of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having an elevated T-beta level is present in any one of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any one of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated T-beta level.

In some embodiments, step (iii) comprises measuring the ICOS and T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having elevated ICOS and T-beta levels is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having elevated ICOS and/or T-beta levels.

In some embodiments, step (iii) comprises measuring the ICOS and T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, step (iv) comprises determining whether a CD4+ T cell population having elevated ICOS and T-beta levels is present in any of the one or more peripheral blood test samples when compared to a control, and step (v) comprises administering to the subject (a) one or more additional doses of the one or more anti-cancer therapies, or (b) an anti-ICOS agonist, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having elevated ICOS and T-beta levels.

In some embodiments, the one or more anti-cancer therapies comprise immunotherapy, such as an anti-CTLA-4 antagonist antibody (e.g., ipilimumab, tremelimumab, or BMS-986249); anti-PD-1 or anti-PD-L1 antagonist antibodies (e.g., avilumab (avelumab), atelizumab, CX-072, pembrolizumab, nivolumab, cimiralizumab, sibradizumab (spartalizumab), tiramizumab (tiselizumab), JNJ-63723283, geminzumab (geniumzumab), AMP-514, age 2034, dewalizumab, or JNC-1); or an anti-ICOS agonist antibody (e.g., JTX-2011, BMS-986226, or GSK 3359609). In some embodiments, the anti-ICOS agonist comprises an anti-ICOS agonist antibody (e.g., JTX-2011, BMS-986226, or GSK 3359609).

In some embodiments, the one or more anti-cancer therapies comprise one or more of the therapies listed in table 2.

In some embodiments, the one or more anti-cancer therapies comprise chemotherapy (e.g., capecitabine, cyclophosphamide, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine, irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, nabumetone (nab-paclitaxel), pemetrexed, vinorelbine, vincristine, erlotinib, afatinib, gefitinib, crizotinib, darafinib, trametinib, vemurafenib, and cobimetanib).

In some embodiments, the one or more anti-cancer therapies comprise radiation therapy.

In some embodiments, step (v) comprises administering to the subject an anti-ICOS antibody agonist if any of the one or more peripheral blood test samples is determined to comprise a population of CD4+ T cells having elevated ICOS and/or T-beta levels.

In some embodiments, the anti-ICOS antibody agonist comprises at least one CDR selected from the group consisting of: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO 10 wherein one or more of the CDRs comprise 1 or 2 amino acid substitutions.

In some embodiments, the anti-ICOS antibody agonist comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the anti-ICOS antibody agonist comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:1, and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the one or more anti-cancer therapies comprise any combination of at least two or at least three of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

In some embodiments, the one or more anti-cancer therapies are administered two, three, four, five, or more times prior to obtaining the one or more peripheral blood test samples.

In some embodiments, obtaining the one or more peripheral blood test samples is performed less than 4 weeks, 3 weeks, 2 weeks, or less than 1 week after the one or more doses of the one or more anti-cancer therapies.

In some embodiments, the dose of the one or more anti-cancer therapies is administered multiple times at regular intervals, e.g., the regular intervals are regular intervals selected from the group consisting of: weekly, biweekly, every three weeks, every four weeks, every six weeks, every nine weeks, and every twelve weeks.

In some embodiments, the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein the test samples are obtained concurrently with the one or more administrations.

In some embodiments, the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein the test samples are obtained during a time period between the plurality of administrations.

In some embodiments, the method further comprises discontinuing administration of the one or more anti-cancer therapies if a population of CD4+ T cells having an elevated ICOS and/or T-beta level compared to a control is not detected in any of the peripheral blood test samples after administration of the one or more anti-cancer therapies for four or more intervals.

In some embodiments, the method further comprises discontinuing administration of the one or more anti-cancer therapies if a peripheral blood test sample is obtained after administration of the one or more anti-cancer therapies for five or more, six or more, seven or more, eight or more, nine or more, or ten or more intervals, based on which it is determined that there is no population of CD4+ T cells having elevated ICOS and/or T-beta levels compared to a control.

In some embodiments, the method further comprises storing a portion of one or more of the peripheral blood test samples.

In some embodiments, a portion of CD4+ T cells having elevated ICOS and/or T-beta levels are isolated from one or more of the peripheral blood test samples and stored under conditions suitable for maintaining the viability of CD4+ T cells.

In some embodiments, the stored CD4+ T cells are stored in cell culture media.

In some embodiments, the stored CD4+ T cells are stored at a concentration greater than 100,000 cells/mL, for example, between 100,000 cells/mL and 1 hundred million cells/mL.

The present invention also provides a suspension of CD4+ T cells obtained according to the methods described herein.

In some embodiments, the control comprises a peripheral blood test sample, optionally obtained from the subject prior to one or more administrations of the one or more anti-cancer therapies to the subject.

In some embodiments, the control comprises a peripheral blood sample obtained from a healthy individual who has not received the one or more anti-cancer therapies.

In some embodiments, determination of ICOS and/or T-beta levels comprises use of an immunoassay, e.g., an immunoassay comprising use of an antibody that binds to the intracellular domain of ICOS to detect ICOS.

In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO 27 and the light chain variable region sequence of SEQ ID NO 31; or the antibody comprises the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

In some embodiments, the antibody cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 27 and the light chain variable region sequence of SEQ ID NO 31; or cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

In some embodiments, the methods further comprise measuring the ICOS and/or T-beta levels of CD8+ T cells present in the one or more peripheral blood test samples, wherein a CD8+ T cell population having elevated ICOS and/or T-beta levels relative to a control is not detected in the sample.

In some embodiments, the cancer is selected from gastric cancer, breast cancer optionally Triple Negative Breast Cancer (TNBC), non-small cell lung cancer (NSCLC), melanoma, Renal Cell Carcinoma (RCC), bladder cancer, endometrial cancer, diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, ovarian cancer, and Head and Neck Squamous Cell Carcinoma (HNSCC).

In some embodiments, the subject is a human patient.

In some embodiments, the population of CD4+ T cells with elevated ICOS and/or T-beta levels comprises a novel population of CD4+ T cells alone induced by one or more anti-cancer therapies.

The invention also includes a method of producing an expanded population of CD4+ T cells with elevated ICOS expression, the method comprising culturing the aforementioned CD4+ T cell suspension under initial culture conditions suitable for expansion of a CD4+ T cell population. These initial conditions suitable for expanding a CD4+ T cell population may comprise contacting the suspension with: for example, CD3 agonists (e.g., OKT 3); one or more of an anti-PD-1 antibody antagonist, an anti-CTLA-4 antibody, and an ICOS agonist; and optionally, selected from the group consisting of IL-2, IL-12 and anti IL-4 group of one or more compounds (e.g., two or more or all three). These methods may further comprise contacting the suspension with, for example, a CD28 agonist. In certain embodiments, the CD3 agonist and the anti-CD 28 agonist are present in a tetrameric antibody complex. In such methods, a suspension of CD4+ T cells is incubated under the initial culture conditions, for example, for a period of time between one and five days (e.g., about 1 day, 2 days, 3 days, 4 days, or 5 days).

In certain embodiments, the method may further comprise incubating the CD4+ T cell suspension under second culture conditions suitable for expansion of the CD4+ T cell population. Here, optionally, the cells are washed prior to applying the second culture conditions. In certain embodiments, the second culture conditions can include, for example, contacting the cell suspension with an anti-PD-1 antibody antagonist, an anti-CTLA-4 antibody, and an ICOS agonist. In certain embodiments, the second culture condition comprises contacting the cell suspension with one or more compounds (e.g., two or more or all three) selected from the group comprising IL-2, IL-12, and anti-IL-4. Additionally or alternatively, the second culturing condition comprises contacting the cell suspension with an anti-CD 28 antibody agonist. Alternatively, in certain embodiments, the second culture condition does not comprise contacting the cell suspension with a CD3 agonist and/or a CD28 agonist. The second culturing condition can be maintained, for example, between 1 day and 5 days (e.g., 1 day, 2 days, 3 days, 4 days, or 5 days).

In another embodiment, the invention features a suspension of cells produced by any of the foregoing culturing methods.

The invention also provides a method for determining whether a subject may benefit from (a) continued treatment with one or more anti-cancer therapies or (b) treatment with an anti-ICOS agonist, the method comprising determining ICOS and/or T-beta levels of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of increased ICOS and/or T-beta levels relative to a control indicates that the subject may benefit from continued treatment optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

In some embodiments, the method comprises determining the ICOS level of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of an increased ICOS level relative to a control indicates that the subject may benefit from continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies do not comprise anti-ICOS antibody agonist treatment.

In some embodiments, the method comprises determining a T-beta level of peripheral CD4+ T cells in a blood sample from the subject, wherein detection of an increased T-beta level relative to a control indicates that the subject may benefit from continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies does not comprise anti-ICOS antibody agonist treatment.

In some embodiments, the method comprises determining ICOS and T-beta levels of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of increased ICOS or T-beta levels relative to a control indicates that the subject may benefit from continued treatment, optionally in combination with an anti-ICOS antibody agonist, or treatment with an anti-ICOS agonist when the one or more anti-cancer therapies do not comprise anti-ICOS antibody agonist treatment.

In some embodiments, the one or more anti-cancer therapies comprise immunotherapy, such as an anti-CTLA-4 antagonist antibody (e.g., ipilimumab, tremelimumab, or BMS-986249); anti-PD-1 or anti-PD-L1 antagonist antibodies (e.g., avilumab (avelumab), atelizumab, CX-072, pembrolizumab, nivolumab, cimiralizumab, sibradizumab (spartalizumab), tiramizumab (tiselizumab), JNJ-63723283, geminzumab (geniumzumab), AMP-514, age 2034, dewalizumab, or JNC-1); or an anti-ICOS agonist antibody (e.g., JTX-2011, BMS-986226, or GSK 3359609). In some embodiments, the anti-ICOS agonist comprises an anti-ICOS agonist antibody (e.g., JTX-2011, BMS-986226, or GSK 3359609).

In some embodiments, the one or more anti-cancer therapies comprise one or more of the therapies listed in table 2.

In some embodiments, the one or more anti-cancer therapies comprise chemotherapy (e.g., capecitabine, cyclophosphamide, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine, irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, nabumetone (nab-paclitaxel), pemetrexed, vinorelbine, vincristine, erlotinib, afatinib, gefitinib, crizotinib, darafinib, trametinib, vemurafenib, and cobimetanib).

In some embodiments, the one or more anti-cancer therapies comprise radiation therapy.

In some embodiments, step (v) comprises administering to the subject an anti-ICOS antibody agonist if any of the one or more peripheral blood test samples is determined to comprise a population of CD4+ T cells having elevated ICOS and/or T-beta levels.

In some embodiments, the anti-ICOS antibody agonist comprises at least one CDR selected from the group consisting of: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO 10 wherein one or more of the CDRs comprise 1 or 2 amino acid substitutions.

In some embodiments, the anti-ICOS antibody agonist comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the anti-ICOS antibody agonist comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:1, and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the one or more anti-cancer therapies comprise any combination of at least two or at least three of: (i) an anti-CTLA-4 antagonist antibody, (ii) an anti-PD-1 or anti-PD-L1 antagonist antibody, (iii) an anti-ICOS agonist antibody, (iv) the therapies of table 2, (v) chemotherapy, and (vi) radiation therapy.

In some embodiments, the one or more anti-cancer therapies are administered two, three, four, five, or more times prior to obtaining the one or more peripheral blood test samples.

In some embodiments, obtaining the one or more peripheral blood test samples is performed less than 4 weeks, 3 weeks, 2 weeks, or less than 1 week after the one or more doses of the one or more anti-cancer therapies.

In some embodiments, the dose of the one or more anti-cancer therapies is administered multiple times at regular intervals, e.g., the regular intervals are regular intervals selected from the group consisting of: weekly, biweekly, every three weeks, every four weeks, every six weeks, every nine weeks, and every twelve weeks.

In some embodiments, the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein the test samples are obtained concurrently with the one or more administrations.

In some embodiments, the obtaining of the one or more peripheral blood test samples comprises obtaining a plurality of peripheral blood test samples, wherein the test samples are obtained during a time period between the plurality of administrations.

In some embodiments, the method further comprises discontinuing administration of the one or more anti-cancer therapies if a population of CD4+ T cells having an elevated ICOS and/or T-beta level compared to a control is not detected in any of the peripheral blood test samples after administration of the one or more anti-cancer therapies for four or more intervals.

In some embodiments, the method further comprises discontinuing administration of the one or more anti-cancer therapies if a peripheral blood test sample is obtained after administration of the one or more anti-cancer therapies for five or more, six or more, seven or more, eight or more, nine or more, or ten or more intervals, based on which it is determined that there is no population of CD4+ T cells having elevated ICOS and/or T-beta levels compared to a control.

In some embodiments, the method further comprises storing a portion of one or more of the peripheral blood test samples.

In some embodiments, a portion of CD4+ T cells having elevated ICOS and/or T-beta levels are isolated from one or more of the peripheral blood test samples and stored under conditions suitable for maintaining the viability of CD4+ T cells.

In some embodiments, the stored CD4+ T cells are stored in cell culture media.

In some embodiments, the stored CD4+ T cells are stored at a concentration greater than 100,000 cells/mL, for example, between 100,000 cells/mL and 1 hundred million cells/mL.

The present invention also provides a suspension of CD4+ T cells obtained according to the methods described herein.

In some embodiments, the control comprises a peripheral blood test sample, optionally obtained from the subject prior to one or more administrations of the one or more anti-cancer therapies to the subject.

In some embodiments, the control comprises a peripheral blood sample obtained from a healthy individual who has not received the one or more anti-cancer therapies.

In some embodiments, determination of ICOS and/or T-beta levels comprises use of an immunoassay, e.g., an immunoassay comprising use of an antibody that binds to the intracellular domain of ICOS to detect ICOS.

In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO 27 and the light chain variable region sequence of SEQ ID NO 31; or the antibody comprises the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

In some embodiments, the antibody cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 27 and the light chain variable region sequence of SEQ ID NO 31; or cross-competes with an antibody comprising the heavy chain variable region sequence of SEQ ID NO 35 and the light chain variable region sequence of SEQ ID NO 39.

In some embodiments, the methods further comprise measuring the ICOS and/or T-beta levels of CD8+ T cells present in the one or more peripheral blood test samples, wherein a CD8+ T cell population having elevated ICOS and/or T-beta levels relative to a control is not detected in the sample.

In some embodiments, the cancer is selected from gastric cancer, breast cancer optionally Triple Negative Breast Cancer (TNBC), non-small cell lung cancer (NSCLC), melanoma, Renal Cell Carcinoma (RCC), bladder cancer, endometrial cancer, diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, ovarian cancer, and Head and Neck Squamous Cell Carcinoma (HNSCC).

In some embodiments, the subject is a human patient.

In some embodiments, the population of CD4+ T cells with elevated ICOS and/or T-beta levels comprises a novel population of CD4+ T cells alone induced by one or more anti-cancer therapies.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIG. 1 is a schematic showing M13 anti-ICOS detection antibody (see, e.g., Table 3 and WO 2017/070423; 2M13) binding to an intracellular epitope of ICOS. Binding of M13 to ICOS did not interfere with the binding of the therapeutic anti-ICOS antibody JTX-2011 to the extracellular region of ICOS.

FIG. 2 is a diagram showing a method of determining whether a sample includes ICOS^{Height of}CD4+Schematic of a method for T cell population.

FIG. 3A is a series of contour plots showing ICOS expression in CD4+ T cells in samples from gastric cancer patients with a demonstrated partial response to monotherapy at 0.3mg/kg (q3w) JTX-2011 over cycle 3-15 (cPR).

Figure 3B is a graph showing the percentage change in size over time of target lesions in samples from gastric cancer patients with a demonstrated partial response to monotherapy with 0.3mg/kg (q3w) JTX-2011 over cycle 3-15 (cPR).

FIG. 4 is a series of contour plots showing ICOS expression in CD4+ T cells in samples from gastric cancer patients with demonstrated partial responses to combination therapy with 0.1mg/kg JTX-2011 and 240mg nivolumab (q3w) over cycle 7-11.

FIG. 5 is a series of contour plots showing ICOS expression in CD4+ T cells in samples from gastric cancer patients with stable disease who received 0.3mg/kg JTX-2011 and 240mg nivolumab in cycles 4-6 in combination therapy (q3 w).

Figure 6 is a series of contour plots showing ICOS expression in CD4+ T cells in a sample from a Triple Negative Breast Cancer (TNBC) patient with stable disease or progressive disease who received a combination therapy of 0.3mg/kg JTX-2011 and 240mg nivolumab (q3 w).

Fig. 7A is a graph showing the number of cancer patients receiving JTX-2011 monotherapy or JTX-2011 and nivolumab combination therapy with CD4+ T cells with and without an increase in ICOS levels (yes and no). Circles represent patients with progressive disease, squares represent patients with stable disease, filled triangles represent patients with unproven partial response, and unfilled triangles represent patients with confirmed partial response.

FIG. 7B is a graph comparing the appearance of ICOS in cancer patients receiving JTX-2011 monotherapy or JTX-2011 and nivolumab combination therapy^hiWaterfall plot of percent change from baseline in target lesions of CD4+ T cell population.

Figure 7C is a graph showing the percentage of cancer patients with Progressive Disease (PD), Stable Disease (SD), unproven Positive Response (PR) and confirmed positive response (cPR) to JTX-2011 monotherapy or JTX-2011 and nivolumab combination therapy with and without CD4+ T cells with increased ICOS expression.

FIG. 8A is a graph showing the increase in ICOS staining 48 hours after dosing relative to 1 hour after dosing in Sa1/N tumor mice receiving a weekly dose of 0.25mG/kg JTX-10110-mG2 a.

FIG. 8B is a contour plot showing ICOS expression in CD4+ T cells from Sa1/N tumor mice receiving a once weekly dose of 0.25mG/kg JTX-10110-mG2a at 48 hours post-dose.

FIG. 9 is a series of contour plots showing ICOS expression in CD4+ T cells in samples from gastric cancer patients with a demonstrated partial response (cPR) to combination therapy with 0.1mg/kg JTX-2011 and 240mg nivolumab over cycles 7-12 (q3 w). ICOS + CD4+ T cells were evaluated for T-beta expression at cycle 12.

FIG. 10 is a series of contour plots showing ICOS and T-beta expression in CD4+ T cells in samples from gastric cancer patients with a demonstrated partial response to 0.3mg/kg (q3w) JTX-2011 monotherapy over cycle 3-15 (cPR).

FIG. 11 is a series of contour plots showing ICOS and T-beta expression in CD4+ T cells in samples from TNBC patients with stable disease treated with a combination therapy of 0.3mg/kg JTX-2011 and 240mg nivolumab over cycles 5-7 (q3 w).

FIG. 12 is a series of contour plots showing ICOS and T-beta expression in CD4+ T cells in samples from TNBC patients with stable disease treated with a combination therapy of 0.3mg/kg JTX-2011 and 240mg nivolumab over cycles 1-9 (q3 w).

FIG. 13 is a graph showing stimulation with tetanus toxoid followed by ICOS^loAnd ICOS^hiA series of graphs of the levels of IFN γ, TNF α and IL-2 in CD4+ T cells. A representative contour plot highlighting two cell populations is shown in the inset. The first of every triplet is IFN γ, the second of every triplet is TNF- α, and the third of every triplet is IL-2.

Fig. 14A is a table showing a summary of ICOS expression of samples detected by flow cytometry profiling.

Fig. 14B is a pair of overlapping histograms showing ICOS levels of CD4+ T cells in NSCLC patients responsive to nivolumab (left panel) and pembrolizumab (right panel) at different time points. The histograms are arranged in chronological order starting from the baseline characteristic of each responder.

FIG. 15A is a graph showing genes that are significantly differentially expressed (FDR adjusted p-value)<0.05) and defines the cross ICOS^hiAnd ICOS^loGene expression heatmap of key components of transcriptional differences of CD4+ T cells.

FIG. 15B is a generalized ICOS^hiAnd ICOS^loTable of major effector pathway regulation in CD4+ T cell population. The approach is shown with overall FDR corrected q values below 0.5.

Figure 15C is a schematic diagram summarizing the allograft rejection pathway.

FIG. 16A shows a uniform ICOS at a later cycle^hiA series of contour plots of Tbet, CD25, FoxP3, and TIGIT expression in T cell populations.

FIG. 16B is a series of graphs showing expression of Lang-3, TIGIT, FoxP3, CD4, and CD8 in gastric cancer patients with cPR at two time points.

Fig. 17 is a graph showing early and late proliferation of CD8+ and CD4+ T cells in subjects with confirmed PR for JTX-2011 treatment. The average of 4 subjects profiled longitudinally is shown.

FIG. 18 is a graph showing polyclonal expansion of TCR pools following treatment with JTX-2011. The average of 22 subjects is shown.

FIGS. 19A and 19B are graphs showing the presence of ICOS in the absence (FIG. 19A) and presence (FIG. 19B)^hiA pair of plots of the frequency of tumor-associated clones and de novo clones in subjects with CD4+ T cells.

FIG. 20A is a flow cytometric display of ICOS of a patient with cPR^hiHistogram of homogeneity of CD4+ T cell population.

Figure 20B is a graph showing antigen-specific responses as measured by ELISPOT of PBMCs isolated from patients with known mutation status. The first was a negative control, the second was a positive control, and the third was KRAS/PTEN/BRAF. The mutations tested were KRAS G12D, PTEN R173C and BRAF E26D. The length of the peptide is 15 amino acids. The mutation is located in the peptide center flanked by 7 amino acids of the wild-type sequence on each side. The three peptides were each combined at 2. mu.g/mL. The positive control was a 2. mu.g/mL CEF peptide library consisting of common CMV, EBV and influenza antigens. The negative control is vehicle.

FIG. 21 is a schematic diagram showing an ICOS^hiGraph of six month median PFS of patients with T cell appearance.

FIG. 22 is a diagram showing that ICOS has not been reached^hiGraph of median OS of patients with T cell appearance.

Detailed Description

Methods of treating cancer are provided. The methods include treating the subject with one or more anti-cancer therapies, and then determining whether a peripheral blood sample of the subject includes CD4+ T cells having elevated ICOS and/or T-beta levels. If so, the subject is either (a) continued to be treated with one or more anti-cancer therapies, optionally in combination with an anti-ICOS agonist antibody, or (b) treated with an anti-ICOS agonist (e.g., an anti-ICOS agonist antibody) without further treatment with one or more anti-cancer therapies. Also provided are methods of determining whether a subject may benefit from continued treatment with one or more anti-cancer therapies or treatment with an anti-ICOS agonist (e.g., an anti-ICOS agonist antibody) based on detection of elevated ICOS and/or T-beta levels in CD4+ T cells from a peripheral blood sample. If such a population of cells is identified in the sample, the subject may benefit from (a) continued treatment with one or more anti-cancer therapies (optionally in combination with an anti-ICOS agonist antibody), or (b) treatment with an anti-ICOS agonist (e.g., an anti-ICOS agonist antibody) without further treatment with one or more anti-cancer therapies.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

All references, including patent applications, patent publications, and Genbank accession numbers, cited herein are hereby incorporated by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

I. Definition of

Unless defined otherwise, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise or is clearly indicated, singular terms shall include the plural and plural terms shall include the singular. For any conflict in definition between various sources or references, the definitions provided herein control.

It is to be understood that the embodiments of the invention described herein include "consisting of" and/or "consisting essentially of" the embodiments.

As used herein, the singular forms "a", "an" and "the" include plural referents unless otherwise indicated.

The use of the term "or" herein is not meant to imply that alternatives are mutually exclusive.

In this application, the use of "or" means "and/or" unless explicitly stated or explained by a person skilled in the art. In the context of several dependent claims, the use of "or" means to recede more than one prior independent or dependent claim.

As understood by those skilled in the art, reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" refers to a linear sequence of nucleotides that make up a nucleic acid molecule or polynucleotide.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conserved in nature) to the native sequence, so long as the protein retains the desired activity. These modifications may be made manually, for example by site-directed mutagenesis, or occasionally, such as by mutation of the host producing the protein or by error due to PCR amplification.

As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN^TM(DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full-length sequences being compared.

Amino acid substitutions may include, but are not limited to, the substitution of one amino acid for another in a polypeptide. Exemplary substitutions are shown in table 1. Amino acid substitutions can be introduced into an antibody of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

TABLE 1

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of members of one of these classes for another.

As used herein, "ICOS" and "inducible T cell costimulation" refer to any native ICOS that results from the expression and processing of ICOS in a cell. Unless otherwise indicated, the term includes ICOS from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of ICOS, such as splice variants or allelic variants. The amino acid sequence of an exemplary human ICOS precursor protein having a signal sequence (amino acids 1-20) is shown in SEQ ID NO 11. The amino acid sequence of an exemplary mature human ICOS is set forth in SEQ ID NO 12. The intracellular portion of ICOS is underlined in SEQ ID NO 11 and 12 in Table 3. The amino acid sequence of an exemplary mouse ICOS precursor protein having a signal sequence (amino acids 1-20) is shown in SEQ ID NO 13. The amino acid sequence of an exemplary mature mouse ICOS is shown in SEQ ID NO 14. The amino acid sequence of an exemplary rat ICOS precursor protein having a signal sequence (amino acids 1-20) is shown in SEQ ID NO 15. The amino acid sequence of an exemplary mature rat ICOS is shown in SEQ ID NO 16. The amino acid sequence of an exemplary cynomolgus monkey ICOS precursor protein having a signal sequence (amino acids 1-20) is shown in SEQ ID NO 17. An exemplary mature cynomolgus monkey ICOS has the amino acid sequence shown in SEQ ID NO 18.

"T-beta", "T-cell specific T-box transcription factor T-Bet" or "T-box 21" as used herein refers to any native T-beta encoded by the TBX21 gene resulting from the expression and processing of T-beta in a cell. Unless otherwise indicated, the term includes T-beta from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of T-beta, such as splice variants or allelic variants. The amino acid sequence of an exemplary human T-beta protein is shown in SEQ ID NO 43. An exemplary mouse T-beta amino acid sequence is shown in SEQ ID NO 44.

The term "specifically binds" to an antigen or epitope is a term well known in the art, and methods of determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and/or with greater affinity with a particular cell or substance than with a replacement cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an ICOS epitope is an antibody that binds to this epitope with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other ICOS epitopes or non-ICOS epitopes. It will also be understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding. "specificity" refers to the ability of a binding protein to selectively bind to an antigen.

As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most preferably at least 99% pure.

As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an antibody, antibody fragment containing an antibody binding region, or scaffold protein) binds. Epitopes generally comprise chemically active surface groups of molecules, such as amino acids, polypeptides or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed by contiguous and/or juxtaposed non-contiguous residues (e.g., amino acid, nucleotide, sugar, or lipid moieties) of the target molecule. Epitopes formed from contiguous residues, also referred to as linear epitopes (e.g., amino acid, nucleotide, sugar or lipid moieties), are typically retained upon exposure to denaturing solvents, whereas epitopes formed from non-contiguous residues (also referred to as non-linear or conformational epitopes) are formed by tertiary folding and are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some examples, the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length.

If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen or to overlapping epitopes. Thus, in some embodiments, an antibody is said to "cross-compete" with another antibody if the antibody specifically interferes with binding of the antibody to the same or an overlapping epitope.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific (such as bispecific T cell cement) and trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen binding activity.

The term antibody includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single chain Fv (scFv), Fab ', di-scFv, sdAb (single domain antibody), and (Fab ')2 (including chemically linked F (ab ') 2). Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each of which has a single antigen binding site; and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces F (ab')2 fragments that have two antigen binding sites and are still capable of cross-linking antigens. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species (such as mouse, human, cynomolgus monkey, etc.). Furthermore, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, if a human-derived form of the antibody is disclosed, one skilled in the art would understand how to convert an antibody based on human sequences into sequences for mice, rats, cats, dogs, horses, etc. Antibody fragments also include the orientation of single chain scFv, tandem di-scFv, diabodies, tandem tri-sdcFvs, minibodies, and the like. Antibody fragments also include nanobodies (sdabs, antibodies with a single monomer domain, such as a pair of heavy chain variable domains, without a light chain). In some embodiments, an antibody fragment may be referred to as being species-specific (e.g., a human scFv or a mouse scFv). This indicates the sequence of at least part of the non-CDR region, not the origin of the construct.

The term "monoclonal antibody" refers to an antibody of a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a monoclonal antibody sample can bind to the same epitope on an antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler and Milstein,1975, Nature 256:495, or can be prepared by recombinant DNA methods, such as described in U.S. Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using techniques such as those described in McCafferty et al, 1990, Nature 348: 552-.

The term "CDR" denotes a complementarity determining region as defined by at least one means of identification by those skilled in the art. In some embodiments, the CDRs may be defined according to any one of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, AbM definitions, contact definitions, and/or a combination of Kabat, Chothia, AbM, and/or contact definitions. Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 50-65 of amino acid residues 31-35B, H2 of H1, and amino acid residues 95-102 of H3. (Kabat et al, Sequences of Proteins of lmmunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD (1991)). AbM definitions may include, for example, at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues H26-H35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3 (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3). The Contact definition may include, for example, at amino acid residues 30-36 of L1, amino acid residues 46-55 of L2, amino acid residues 89-96 of L3, amino acid residues 30-35 of H1, amino acid residues 47-58 of H2, and amino acid residues 93-101 of H3 (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3). Chothia definitions may include, for example, at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 26-32.. 34 of H1, amino acid residues 52-56 of H2, and amino acid residues 95-102 of H3 (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3). Except for V_HIn (3), the CDR generally comprises amino acid residues forming a hypervariable loop, in addition to CDR 1. The different CDRs in an antibody may be grouped by their appropriate number and chain typeType, including but not limited to: a) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3; b) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH 3; c) LCDR-1, LCDR-2, LCDR-3, HCDR-1, HCDR-2, and HCDR-3; or d) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR 3; and so on. The term "CDR" as used herein also encompasses HVRs or "hypervariable regions", including hypervariable loops. Exemplary hypervariable loops occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987)).

As used herein, the term "heavy chain variable region" refers to a region comprising at least three heavy chain CDRs. In some embodiments, the heavy chain variable region comprises three CDRs and at least FR2 and FR 3. In some embodiments, the heavy chain variable region comprises at least heavy chain HCDR1, Framework Region (FR)2, HCDR2, FR3, and HCDR 3. In some embodiments, the heavy chain variable region further comprises at least a portion of FR1 and/or at least a portion of FR 4.

The term "heavy chain constant region" as used herein refers to a region comprising at least three heavy chain constant domains CH1, CH2, and CH 3. Of course, deletions and alterations that do not alter function within the domain are encompassed within the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, the antibody comprising a mu constant region is an IgM antibody, and the antibody comprising an epsilon constant region is an IgE antibody. Certain isoforms may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising γ)₁Constant region), IgG2 (comprising γ)₂Constant region), IgG3 (comprising γ)₃Constant region) and IgG4 (comprising γ)₄Constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising alpha)₁Constant region) and IgA2 (comprising a)₂Constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM 2.

As used herein, the term "heavy chain" refers to a polypeptide comprising at least one heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

As used herein, the term "light chain variable region" refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region comprises three CDRs and at least FR2 and FR 3. In some embodiments, the light chain variable region comprises at least light chain LCR1, Framework Region (FR)2, LCD2, FR3, and LCD 3. For example, the light chain variable region may comprise light chain CDR1, Framework Region (FR)2, CDR2, FR3 and CDR 3. In some embodiments, the light chain variable region further comprises at least a portion of FR1 and/or at least a portion of FR 4.

The term "light chain constant region" as used herein refers to a region comprising a light chain constant domain C_LThe area of (a). Non-limiting exemplary light chain constant regions include λ and K. Of course, deletions and alterations that do not alter function within the domain are encompassed within the term "light chain constant region" unless otherwise specified.

As used herein, the term "light chain" refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. As used herein, the term "full length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

An "acceptor human framework" for the purposes herein is a framework derived from a human immunoglobulin framework or human consensus framework comprising a light chain variable domain (V)_L) Framework or heavy chain variable domains (V)_H) Framework of the amino acid sequence of the framework, as defined below. Acceptor human frameworks derived from human immunoglobulin frameworks or human consensus frameworks may comprise their same amino acid sequence, or they may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, V_LAcceptor human framework and V_LThe human immunoglobulin framework sequences or human consensus framework sequences are identical in sequence.

"affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD). Affinity can be determined by common methods known in the art (e.g., ELISA KD, KinExA, biolayer interferometry (BLI) and/or surface plasmon resonance devices (such asDevices), including those described herein).

As used herein, the term "KD", or "KD value" refers to the equilibrium dissociation constant of an antibody-antigen interaction.

The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo, or provided or enabled by recombinant means). Biological properties include, but are not limited to, binding to receptors, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and enzymatic activity. In some embodiments, the biological activity of the ICOS protein includes, for example, co-stimulation of T cell proliferation and cytokine secretion associated with Th1 and Th2 cells; modulating Treg cells; effects on T cell differentiation, including regulation of transcription factor gene expression; induces signaling through PI3K and the AKT pathway; and mediate ADCC.

As used herein, the term "substantially similar" or "substantially the same" means a sufficiently high degree of similarity between two or more numerical values such that one of skill in the art would consider the difference between the two or more values to have little or no biological and/or statistical significance within the context of the biological feature measured by the value. In some embodiments, two or more substantially similar values differ by no more than about any of 5%, 10%, 15%, 20%, 25%, or 50%.

As used herein, the phrase "substantially different" means a sufficiently high degree of difference between two numerical values such that one of skill in the art would consider the difference between the two values to have statistical significance in the context of the biological feature measured by the values. In some embodiments, two substantially different values differ by more than any of about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%.

As used herein, the phrase "substantially reduced" means a sufficiently high degree of reduction between a numerical value and a reference numerical value such that one of skill in the art would consider the difference between the two values to have statistical significance in the context of the biological characteristic measured by the value. In some embodiments, the substantially reduced number is reduced by greater than any one of about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to the reference value.

As used herein, the phrase "substantially increases" means a sufficiently high degree of increase between a numerical value and a reference numerical value such that one of skill in the art would consider the difference between the two values to have statistical significance in the context of the biological characteristic measured by the value. In some embodiments, the substantially increased value is increased by greater than any one of about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to the reference value.

As used herein, the term "isolated" refers to a molecule that has been separated from at least some components that are typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is isolated from at least some components of the cell in which it is produced. Where the cell secretes the polypeptide after expression, physically separating the supernatant containing the polypeptide from the cell producing the polypeptide is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide (e.g., genomic or mitochondrial DNA in the case of a DNA polynucleotide) that is typically found in nature, or is separated from at least some of the components of the cell in which it is produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated".

The terms "individual," "patient," or "subject" are used interchangeably herein and refer to an animal, such as a mammal. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, apes, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, indicating the fact that the subject has been identified as having a treatment-related disorder or as being at sufficient risk of having the disorder.

As used herein, the term "sample" or "patient sample" refers to a composition obtained or derived from a subject of interest, which composition contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "test sample" and variations thereof refers to any sample obtained from a subject of interest that would be expected or known to contain the cellular and/or molecular entities to be characterized. By "tissue or cell sample" is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be blood (e.g., peripheral blood) or any blood component; solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells at any time during pregnancy or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. Tissue samples may contain compounds that do not naturally mix with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. In some embodiments, the sample comprises peripheral blood obtained from the subject or patient, which comprises CD4+ cells. In some embodiments, the sample comprises CD4+ cells isolated from peripheral blood.

As used herein, "control," "control sample," "reference," or "reference sample" refers to any sample, standard, or level used for comparison purposes. Controls or references can be obtained from healthy and/or non-diseased samples. In some examples, a control or reference can be obtained from an untreated sample or untreated patient. In some examples, the reference is obtained from an individual sample of a subject who is not diseased or treated. In some examples, the reference is obtained from one or more healthy individuals who are not the subject or patient. In some embodiments, a control sample, reference cell, or reference tissue is obtained from a patient or subject at a time point prior to one or more administrations of a treatment (e.g., one or more anti-cancer treatments) or prior to undergoing any of the methods of the invention.

As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and/or desired. In some embodiments, the disease or disorder is cancer.

As used herein, "cancer" and "tumor" are interchangeable terms, and refer to the growth or proliferation of any abnormal cell or tissue in an animal. As used herein, the terms "cancer" and "tumor" encompass solid cancers and hematologic/lymphoid cancers, and also malignant, pre-malignant, and benign growths, such as dysplasia. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific non-limiting examples of such cancers include gastric cancer, breast cancer (e.g., Triple Negative Breast Cancer (TNBC)), non-small cell lung cancer (NSCLC), squamous cell cancer, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, lung adenocarcinoma, lung squamous cancer, peritoneal cancer, hepatocellular cancer, gastrointestinal tract cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial or uterine cancer (including endometrial carcinoma of the uterus), salivary gland cancer, kidney cancer (kidney cancer), kidney cancer (renal cancer), liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatoma), brain cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, melanoma, and various types of head and neck cancer. These and other cancers may be treated or analyzed according to the methods of the invention.

As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. As used herein, "treatment" encompasses any administration or use of a therapeutic agent for a disease in a mammal (including a human). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of disease, preventing or delaying the spread of disease (e.g., metastasis, e.g., to the lungs or lymph nodes), preventing or delaying the recurrence of disease, delaying or slowing the progression of disease, ameliorating the disease state, inhibiting disease or disease progression, inhibiting or slowing disease or progression thereof, arresting the progression thereof, and relieving (whether partial or total). "treating" also encompasses reducing the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require one hundred percent removal of all aspects of the disorder.

By "ameliorating" is meant reducing or ameliorating one or more symptoms, e.g., as compared to not administering an anti-cancer therapy. "improving" also includes shortening or reducing the duration of symptoms.

In the context of cancer, the term "treatment" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

As used herein, "prevention" includes providing prevention against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease. The terms "reduce", "inhibit" or "prevent" do not mean or require complete prevention at all times, unless otherwise indicated.

"predetermined cut-off value" and "predetermined level" generally refer to a measured cut-off value used to assess the diagnostic/prognostic/therapeutic efficacy outcome by comparing the measured outcome to a predetermined cut-off value/level, wherein the predetermined cut-off value/level has been linked or correlated to various clinical parameters (e.g., severity of disease, progression/non-progression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well understood that the cut-off value may vary depending on the nature of the immunoassay (e.g., the antibody employed, etc.). Furthermore, it is well within the skill of one of ordinary skill in the art to adapt the disclosure herein to other immunoassays to obtain immunoassay-specific cut-offs for those other immunoassays based on the present disclosure. While the exact value of the predetermined cut-off/level may vary between assays, the correlations described herein (if any) are generally applicable.

In some embodiments, the terms "increased ICOS level," increased ICOS, "" ICOS^{Height of}And ICOS^hiBy "is meant an increase in ICOS levels in cells (e.g., CD4+ T cells) of a subject in a peripheral blood sample of the subject, e.g., after treatment of the subject with one or more anti-cancer therapies. The increased level can be determined relative to a control, which can be, for example, a peripheral blood sample from the subject being treated, but can be prior to any treatment with the one or more anti-cancer therapies, either entirely or prior to treatment with a second or further cycle of the one or more anti-cancer therapies. Alternatively, the control may be the level of a matching sample (e.g., a peripheral blood sample) from a healthy individual. In some embodiments, the level of ICOS is determined as the level of protein expressed, which in some embodiments may be detected using an antibody directed against the intracellular portion of ICOS. In some embodiments, detection using such antibodies is accomplished by using flow cytometry. In some embodiments, the mean fluorescence is strongAn increase in degree (MFI) of at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or 15-fold) relative to a control indicates that an increase in ICOS levels is detected. In some embodiments, detection of an increase in ICOS level in at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of CD4+ T cells in a peripheral blood sample is indicative of the subject having an ICOS hi sample. In some embodiments, an at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or 15-fold) increase in Mean Fluorescence Intensity (MFI) in at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of CD4+ T cells in the peripheral blood sample relative to a control indicates that an increase in ICOS levels is detected. In some embodiments, an elevated ICOS level refers to an increase in total ICOS expression level (e.g., mRNA level or protein level) in CD4+ T cells in a peripheral blood test sample by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, or more, relative to a control sample. In some embodiments, an elevated ICOS level refers to an increase in total ICOS expression level (e.g., mRNA level or protein level) in CD4+ T cells in a peripheral blood sample by at least about 1.1x, 2x, 3x, 4x, 5x, 10x, 15x, 20x, 30x, 40x, 50x, 100x, 500x, 1000x, or more fold relative to a control sample.

In some embodiments, the terms "elevated T-beta level", "elevated T-beta", "T-beta^{Height of}"and" T-beta^hi"refers to an increase in T-beta levels in a subject's cells (e.g., CD4+ T cells) in a subject's peripheral blood sample, e.g., after treatment of the subject with one or more anti-cancer therapies. The increased level can be determined relative to a control, which can be, for example, a peripheral blood sample from the subject being treated, but can be prior to any treatment with the one or more anti-cancer therapies, either entirely or prior to treatment with a second or further cycle of the one or more anti-cancer therapies. Alternatively, the control may be the level of a matching sample (e.g., a peripheral blood sample) from a healthy individual. In some embodiments, the protein is expressed asLevels determine the level of T-beta, which in some embodiments can be detected using antibodies against T-beta. In some embodiments, detection using such antibodies is accomplished by using flow cytometry. In some embodiments, an increase in Mean Fluorescence Intensity (MFI) of at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or 15-fold) relative to a control indicates that an increase in T-beta levels is detected. In some embodiments, detection of an increase in T-beta levels in at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of CD4+ T cells in the peripheral blood sample indicates that the subject has a T-beta hi sample. In some embodiments, an at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, or 15-fold) increase in Mean Fluorescence Intensity (MFI) in at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of CD4+ T cells in the peripheral blood sample relative to a control indicates that an increase in T-beta levels is detected. In some embodiments, elevated T-beta levels refers to an increase in total T-beta expression level (e.g., mRNA level or protein level) in CD4+ T cells in the peripheral blood test sample by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more relative to a control sample. In some embodiments, an elevated level of T-beta refers to an increase in total T-beta expression level (e.g., mRNA level or protein level) in CD4+ T cells in a peripheral blood sample by at least about 1.1x, 2x, 3x, 4x, 5x, 10x, 15x, 20x, 30x, 40x, 50x, 100x, 500x, 1000x, or more, relative to a control sample.

The term "inhibition" or "inhibition" refers to the reduction or cessation of any phenotypic feature, or the reduction or cessation of the incidence, extent or likelihood of said feature. By "reduce" or "inhibit" is meant reduce, reduce or prevent activity, function and/or amount as compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 20% or greater. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 50% or greater. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time relative to a control dose (such as a placebo) over the same period of time.

As used herein, "delaying the progression of a disease" means delaying, impeding, slowing, delaying, stabilizing, inhibiting, and/or delaying the progression of a disease (e.g., cancer). The delay may be of varying lengths of time depending on the medical history and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, as the individual will not suffer from the disease. For example, advanced cancers (such as the development of metastases) may be delayed.

As used herein, "inhibiting" a function or activity refers to decreasing the function or activity when compared to the same other condition except for the condition or parameter of interest, or alternatively as compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of a tumor in the absence of the antibody.

The "therapeutically effective amount" of a substance/molecule, agonist or antagonist may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are counteracted by a therapeutically beneficial effect. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and/or prophylactic result. The therapeutically effective amount of a treatment of the invention can be measured by various endpoints commonly used in evaluating cancer treatments, including, but not limited to: prolonged survival (including OS and PFS); producing objective responses (including CR or PR); tumor regression, tumor weight or size reduction, longer time to disease progression, increased survival duration, longer PFS, increased OS rate, increased duration of response, improved quality of life, and/or improved cancer signs or symptoms.

As used herein, the term "progressive disease" (PD) refers to an increase in the sum of target lesion diameters of at least 20%, based on the minimum sum of the study (including the sum of the baselines if the baseline is the minimum of the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered to be progressing.

As used herein, the term "partial response" (PR) refers to a reduction in the sum of the diameters of the target lesions of at least 30%, with baseline sum diameter as the benchmark.

As used herein, the term "complete response" (CR) refers to the disappearance of all target lesions, wherein the short axis of any target lymph node is reduced to <10 mm.

As used herein, the term "stable disease" (SD) refers to neither sufficient shrinkage to satisfy PR nor sufficient increase to satisfy PD, based on the smallest sum of diameters at the time of study.

As used herein, the term "optimal overall response" (BOR) is the optimal response recorded from the start of study treatment up to the earliest objective progression or the start of a new anti-cancer therapy, taking into account any confirmation requirements. The optimal overall response assignment for a patient will depend on the discovery of targeted and non-targeted diseases, and will also take into account the appearance of new lesions. The optimal overall response is calculated via an algorithm using the assessed responses provided by the investigator during the course of the experiment.

As used herein, the term "non-evaluable" (NE) refers to when an incomplete radiological assessment of a target lesion is performed or a change in the ability of the measurement method to affect a reliable response assessment compared to baseline.

As used herein, the term "objective response rate" (ORR) is equal to the proportion of patients who achieve the best overall response to partial or complete response (PR + CR) according to RECIST 1.1.

As used herein, the term "overall survival" (OS) refers to the percentage of patients that remain viable for a defined period of time (such as 1 year, 5 years, etc.) from the time of diagnosis or treatment. Overall survival was assessed by the Kaplan-Meier method and a 95% Confidence Interval (CI) was provided for the median OS for each treatment group.

As used herein, the term "progression free survival" (PFS) refers to a patient remaining viable without cancer progression or worsening. PFS can be defined as the time from selection of treatment until the first objective progression radiographic material as defined by RECIST (version 1.1) or death for any reason.

By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art used with therapeutic agents, together constituting a "pharmaceutical composition" for administration to a subject. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and are compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation employed.

"sterile" formulations are sterile or substantially free of viable microorganisms and spores thereof.

Administration "in combination with one or more additional therapeutic agents" includes simultaneous (concurrent) and sequential or sequential administration in any order.

The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least a portion of the administrations overlap in time or wherein the administration of one therapeutic agent falls within a short period of time (e.g., within a day) relative to the administration of the other therapeutic agent. For example, two or more therapeutic agents are administered at intervals of no more than about a particular number of minutes.

The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents in which the administration of one or more agents continues after the administration of one or more other agents is discontinued or in which the administration of one or more agents begins before the administration of one or more other agents. For example, two or more therapeutic agents are administered at intervals of more than about a particular number of minutes.

As used herein, "in conjunction with … …" means that another treatment modality is administered in addition to one treatment modality. Thus, "in conjunction with … …" refers to the administration of one treatment modality before, during, or after the administration of other treatment modalities to an individual.

The terms "label" and "detectable label" mean a moiety attached to a polynucleotide or polypeptide such that a reaction (e.g., polynucleotide amplification or antibody binding) is detectable. A polynucleotide or polypeptide comprising a label may be referred to as a "detectably labeled". Thus, the term "labeled binding protein" refers to a protein incorporating a label that provides identification of the binding protein. The terms "labeled oligonucleotide", "labeled primer", "labeled probe", and the like refer to a polynucleotide incorporating a label that provides identification of the nucleic acid comprising or hybridized to the labeled oligonucleotide, primer, or probe. In some embodiments, the label is a detectable label that can produce a signal that can be detected by visual or instrumental means, for example, a polypeptide that incorporates a radiolabeled amino acid or is attached to a biotin moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Examples of markers include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,³H,¹⁴C,³⁵S,⁹⁰Y,⁹⁹Tc,¹¹¹ln,¹²⁵I,¹³¹I,¹⁷⁷Lu,¹⁶⁶Ho,or ¹⁵³sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphorus), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); a chemiluminescent label; a biotinyl group; a predetermined polypeptide epitope recognized by a secondary reporter molecule (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating moieties such as acridinium compounds; and moieties that produce fluorescence, such as fluorescein. In some embodiments, the moiety may itself be detectably labeled, but may become detectable upon reaction with another moiety.

The term "conjugate" refers to an antibody chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" includes chemical compounds, mixtures of chemical compounds, biological macromolecules or extracts made from biological materials. In some embodiments, the therapeutic or cytotoxic agent includes, but is not limited to, pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide (teniposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. When used in the context of an immunoassay, the conjugate antibody may be a detectably labeled antibody that serves as a detection antibody.

As used herein, the term "flow cytometry" generally refers to a technique for characterizing biological particles (such as whole cells or cellular components) by flow cytometry. Methods for performing Flow Cytometry on immune cell samples are well known in the art (see, e.g., Jarosszeski et al, Method in Molecular Biology (1998), Vol. 91: Flow Cytometry Protocols, Humana Press; Longobi Givan, (1992) Flow Cytometry, First Principles, Wiley Liss). All known forms of flow cytometry are intended to be included, particularly Fluorescence Activated Cell Sorting (FACS) in which fluorescently labeled molecules are evaluated by flow cytometry.

The term "amplification" refers to the process of producing one or more copies of a nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., PCR).

As used herein, the technique of "polymerase chain reaction" or "PCR" generally refers to a procedure in which a specific region of a nucleic acid, such as RNA and/or DNA, is amplified, for example, as described in U.S. patent No. 4,683,195. Typically, oligonucleotide primers are designed to hybridize to opposite strands of the template to be amplified, separated by a desired distance. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like.

"quantitative real-time PCR" or "qRT-PCR" refers to a form of PCR in which PCR is performed such that the amount or relative amount of amplified product can be quantified. This technology has been described in various publications including Cronin et al, am.J. Pathol.164(l):35-42 (2004); and Ma et al, Cancer Cell 5: 607-.

The terms "target sequence", "target nucleic acid" or "target nucleic acid sequence" generally refer to a polynucleotide sequence of interest, e.g., a polynucleotide sequence targeted for amplification using, e.g., qRT-PCR.

The term "detecting" includes any means of detection, including direct and indirect detection.

Methods of treatment

The present invention provides methods of treating cancer in a patient in need of such treatment. The method comprises (i) administering one or more doses of one or more anti-cancer therapies to the patient, (ii) obtaining one or more peripheral blood test samples from the patient, (iii) measuring the ICOS and/or T-beta levels of CD4+ T cells present in the one or more peripheral blood test samples, (iv) determining whether a CD4+ T cell population having an elevated ICOS and/or T-beta level is present in any of the one or more peripheral blood test samples when compared to a control, and (v) administering to the patient (a) one or more additional doses of the one or more anti-cancer therapies, or (b) anti-ICOS agonism in the absence of one or more additional doses of the one or more anti-cancer therapies, if any of the one or more peripheral blood test samples is determined to comprise a CD4+ T cell population having an elevated ICOS and/or T-beta level An agent (e.g., an anti-ICOS agonist antibody). Optionally, when the one or more anti-cancer therapies are not anti-ICOS agonist antibody therapies, an anti-ICOS agonist antibody is also administered to a patient determined to have CD4+ T cells with elevated ICOS and/or T-beta levels and being administered one or more additional doses of the one or more anti-cancer therapies.

The present invention also provides methods for determining whether a subject may benefit from continued treatment with one or more anti-cancer therapies. The method comprises determining ICOS and/or T-beta levels of peripheral CD4+ T cells of a blood sample of the subject, wherein detection of increased ICOS and/or T-beta levels relative to a control indicates that the subject is likely to benefit from (a) the continuing treatment, optionally in combination with treatment with an anti-ICOS antibody agonist when the one or more anti-cancer therapies do not include anti-ICOS antibody agonist treatment; or (b) an anti-ICOS agonist (e.g., an anti-ICOS agonist antibody) treatment in the absence of continued treatment with the one or more anti-cancer therapies.

A patient that can be treated as described herein is a patient with cancer. The type of cancer may be any type of cancer listed herein or otherwise known in the art. Exemplary types of cancer include, but are not limited to, gastric cancer, breast cancer (e.g., Triple Negative Breast Cancer (TNBC)), lung cancer (e.g., non-small cell lung cancer (NSCLC)), melanoma, Renal Cell Carcinoma (RCC), bladder cancer, endometrial cancer, diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, ovarian cancer, and Head and Neck Squamous Cell Carcinoma (HNSCC). For additional cancer types that can be treated according to the methods of the invention, see also the definition of cancer above.

Patients that may be treated as described herein include patients who have not previously received an anti-cancer therapy, as well as patients who have previously received (e.g., 1, 2,3, 4, 5, or more) one or more (e.g., 1, 2,3, 4, 5, or more) doses or cycles of an anti-cancer therapy.

Any of the anti-cancer therapies listed herein and other anti-cancer therapies known in the art can be used in conjunction with the methods of the present invention. In some embodiments, the one or more anti-cancer therapies are two or more anti-cancer therapies. In some embodiments, the one or more anti-cancer therapies are three or more anti-cancer therapies. Specific non-limiting examples of anti-cancer therapies that may be used in the present invention are provided below, including, for example, immunotherapy, chemotherapy, cancer vaccines, and the like.

In some embodiments, the one or more anti-cancer therapies are administered once prior to obtaining the one or more peripheral blood test samples from the patient. In some embodiments, the one or more anti-cancer therapies are administered more than once prior to obtaining the one or more peripheral blood test samples from the patient. In some embodiments, the one or more anti-cancer therapies are administered two or more times (e.g., three or more times, four or more times, or five or more times) prior to obtaining the one or more peripheral blood test samples from the patient.

In some embodiments, the one or more anti-cancer therapies are administered to the patient multiple times at regular intervals. These multiple administrations may also be referred to as administration cycles or therapy cycles. In some embodiments, the one or more anti-cancer therapies are administered to the patient for more than two cycles, more than three cycles, more than four cycles, more than five cycles, more than ten cycles, more than fifteen cycles, or more than twenty cycles.

In some embodiments, the regular interval is a weekly dose, every two weeks dose, every three weeks dose, every four weeks dose, every five weeks dose, every six weeks dose, every seven weeks dose, every eight weeks dose, every nine weeks dose, every ten weeks dose, every eleven weeks dose, or every twelve weeks dose.

In some cases, the one or more peripheral blood test samples are obtained from the patient less than four weeks (e.g., less than three weeks, less than two weeks, or less than one week) after administration of the one or more anti-cancer therapies.

In some embodiments, the one or more peripheral blood test samples are obtained as a plurality of samples obtained at a time concurrent with the one or more administrations. In some embodiments, the one or more peripheral blood test samples are obtained as a plurality of samples obtained at times between administrations.

In some embodiments, the control sample is a peripheral blood sample obtained from the same patient prior to administration of the first dose of the one or more anti-cancer therapies (e.g., prior to the first cycle). In some embodiments, the control sample is a peripheral blood sample obtained from the same patient prior to administration of a second or additional (e.g., third, fourth, fifth, or additional) dose of the one or more anti-cancer therapies prior to collection of the peripheral blood test sample. In some embodiments, the control sample is a peripheral blood sample obtained from a healthy patient who has not received an anti-cancer therapy. In some embodiments, the control is a level known or determined to correspond to a level in a control sample as described herein.

In some embodiments, the method further comprises storing a portion of the one or more peripheral blood test samples. In some embodiments, wherein a portion of CD4+ T cells from a peripheral blood test sample are determined to have elevated ICOS and/or T-beta levels relative to CD4+ T cells from a control sample, the method further involves isolating CD4+ T cells having elevated ICOS and/or T-beta levels and storing the CD4+ T cells under conditions suitable to maintain viability of the CD4+ T cells. Any storage method known in the art suitable for maintaining CD4+ T cell viability may be used. In some embodiments, the stored CD4+ T cells are stored in cell culture media. In some embodiments, the stored CD4+ T cells are stored at a concentration greater than 100,000 cells/mL. In some embodiments, the stored CD4+ T cells are stored at a concentration between 100,000 cells/mL and 1 hundred million cells/mL. Thus, in some embodiments, the invention provides the obtained suspension of stored CD4+ T cells.

In some embodiments, the method further comprises administering a therapeutic anti-ICOS agonist antibody to the patient if any of the one or more peripheral blood test samples is determined to comprise a population of CD4+ T cells with elevated ICOS and/or T-beta levels. In some embodiments, the detected population of CD4+ T cells with elevated ICOS and/or T-beta levels is or comprises a new population of isolated CD4+ T cells induced by the one or more anti-cancer therapies. Information on therapeutic anti-ICOS agonist antibodies is provided below.

In some embodiments, the method further comprises discontinuing administration of the one or more anti-cancer therapies if a population of CD4+ T cells with elevated ICOS and/or T-beta levels relative to a control sample is not observed after a predetermined number of administration cycles. The predetermined number of administration cycles can be four or more cycles (e.g., five or more cycles, six or more cycles, or seven or more cycles, eight or more cycles, nine or more cycles, or ten or more cycles). In some embodiments, the methods further comprise discontinuing administration of the one or more anti-cancer therapies prior to a predetermined number of cycles of administration (e.g., four or more cycles) if a population of CD4+ T cells having an elevated ICOS and/or T-beta level relative to a control sample is not observed, and optionally the patient is determined to have progressive disease by conventional methods known in the art (e.g., progressive disease identified by radiographic progression according to RECIST 1.1 criteria; see, e.g., the criteria listed above).

Exemplary anti-cancer therapies for use in the methods of the invention

As an example, any of the anti-cancer therapies listed herein or otherwise known in the art can be used in conjunction with the methods described herein. Exemplary anti-cancer therapies are described below.

a. Immunotherapy

In some embodiments, the one or more anti-cancer therapies are immunotherapy. The interaction between cancer and the immune system is complex and multifaceted. See de Visser et al, nat. rev. cancer (2006)6: 24-37. While many cancer patients appear to develop anti-tumor immune responses, cancer also develops strategies to escape epidemic detection and destruction. Recently, immunotherapy for the treatment and prevention of cancer and other disorders has been developed. Immunotherapy offers cell-specific advantages that other treatments lack. As such, methods for enhancing the efficacy of immune-based therapies may be clinically beneficial.

i. Therapeutic anti-ICOS antibodies

Therapeutic anti-ICOS antibodies that may be used in the present invention include, but are not limited to, humanized antibodies, chimeric antibodies, human antibodies, and antibodies comprising any of the heavy and/or light chain CDRs discussed herein. In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the anti-ICOS antibody is an anti-ICOS agonist antibody. See WO 2016/154177 and WO 2017/070423, each of which is specifically incorporated herein by reference.

In some embodiments, the therapeutic anti-ICOS agonist antibody comprises at least one, two, three, four, five, or all six CDRs selected from: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10. In various embodiments, one or more of the CDRs comprise a substitution or deletion that does not disrupt specific binding to ICOS. In some embodiments, one or more of the CDRs comprise 1, 2,3 or more substitutions, which may optionally include substitutions of conserved amino acids. In some embodiments, one or more of the CDRs comprise 1, 2,3 or more deletions.

In some embodiments, the therapeutic anti-ICOS antibody comprises six CDRs comprising: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the therapeutic anti-ICOS antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the therapeutic anti-ICOS antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the therapeutic anti-ICOS antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region; and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. As used herein, a single chain fv (scfv) or any other antibody comprising a single polypeptide chain including, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have one heavy chain and one light chain. In some embodiments, the heavy chain is a region of the anti-ICOS antibody comprising three heavy chain CDRs. In some embodiments, the light chain is a region of the therapeutic anti-ICOS antibody comprising three light chain CDRs.

In some embodiments, the therapeutic anti-ICOS antibody comprises at least one, at least two, or all three VH CDR sequences selected from: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the therapeutic antibody comprises at least one, at least two, or all three VL CDR sequences selected from: (a) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (b) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (c) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the therapeutic anti-ICOS antibody comprises (I) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; and (II) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from: (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the therapeutic anti-ICOS antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 3. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-ICOS antibody comprising the sequence retains the ability to bind ICOS. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO 3 have been substituted, inserted and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs (i.e., in the FRs). Optionally, the therapeutic anti-ICOS antibody comprises the VH sequence of SEQ ID NO 3, including post-translational modifications of that sequence.

In some embodiments, the VH comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, therapeutic anti-ICOS antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 4. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-ICOS antibody comprising that sequence retains the ability to bind ICOS. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO 4 have been substituted, inserted and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs (i.e., in the FRs). Optionally, the therapeutic anti-ICOS antibody comprises the VL sequence of SEQ ID NO. 4, including post-translational modifications of the sequence.

In some embodiments, the VL comprises: (a) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (b) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (c) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, a therapeutic anti-ICOS antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 3, and a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 4. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, and a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-ICOS antibody comprising the sequence retains the ability to bind ICOS. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO 3 have been substituted, inserted and/or deleted. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO 4 have been substituted, inserted and/or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs (i.e., in the FRs). Optionally, the therapeutic anti-ICOS antibody comprises the VH sequence of SEQ ID NO. 3 and the VL sequence of SEQ ID NO. 4, including post-translational modifications of one or both sequences.

In some embodiments, the therapeutic anti-ICOS antibody comprises (I) a VH domain comprising: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO. 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO 6; and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO. 7; and (II) a VL domain comprising: (d) LCDR1 comprising the amino acid sequence of SEQ ID NO. 8; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO 9; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO 10.

In some embodiments, the therapeutic anti-ICOS antibody comprises a VH as in any embodiment provided herein and a VL as in any embodiment provided herein. In some embodiments, the antibody comprises the VH and VL sequences in SEQ ID NO 3 and SEQ ID NO 4, respectively, including post-translational modifications of those sequences.

In some embodiments, the therapeutic anti-ICOS antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 1, or a variant thereof.

In some embodiments, the therapeutic anti-ICOS antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 2, or a variant thereof.

In some embodiments, the therapeutic anti-ICOS antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 1 and a light chain comprising the amino acid sequence of SEQ ID NO. 2, or variants thereof.

In some embodiments, the therapeutic anti-ICOS antibody comprises six CDRs as described above, and binds ICOS. In some embodiments, the therapeutic anti-ICOS antibody comprises six CDRs as described above, binds to ICOS and increases the number of Teff cells and/or activates Teff cells and/or decreases the number of Treg cells and/or increases the ratio of Teff cells to Treg cells in a mammal (such as a human). In some embodiments, the Treg cells are CD4+ FoxP3+ T cells. In some embodiments, the Teff cells are CD8+ T cells. In some embodiments, Teff cells are CD4+ FoxP3-T cells and/or CD8+ T cells.

Exemplary therapeutic anti-ICOS antibodies include, but are not limited to, JTX-2011 (Vopratelimab; Jounce Therapeutics; US 2016/0304610; WO 2016/154177; WO 2017/070423) and BMS-986226(Bristol-Myers Squibb).

In general, therapeutic anti-ICOS antibodies can be administered in amounts ranging from about 10. mu.g/kg body weight to about 100mg/kg body weight per dose. In some embodiments, the therapeutic anti-ICOS antibody can be administered in an amount ranging from about 50 μ g/kg body weight to about 5mg/kg body weight per dose. In some embodiments, the therapeutic anti-ICOS antibody can be administered in an amount ranging from about 100 μ g/kg body weight to about 10mg/kg body weight per dose. In some embodiments, the therapeutic anti-ICOS antibody can be administered in an amount ranging from about 100 μ g/kg body weight to about 20mg/kg body weight per dose. In some embodiments, the therapeutic anti-ICOS antibody can be administered in an amount ranging from about 0.5mg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, the anti-ICOS antibody can be administered in an amount ranging from about 0.05mg/kg body weight to about 10mg/kg body weight per dose. In some embodiments, the anti-ICOS antibody can be administered in an amount within the range of about 5mg/kg body weight or less, e.g., less than 4mg/kg, less than 3mg/kg, less than 2mg/kg, or less than 1mg/kg of antibody. In specific examples, the therapeutic anti-ICOS antibody is administered at 0.1mg/kg, 0.3mg/kg, or 1.0mg/kg once every 3, 6, 9, or 12 weeks.

anti-CTLA-4 antagonist antibodies

In some embodiments, the one or more anti-cancer therapies are anti-CTLA-4 antagonist antibodies. An anti-CTLA-4 antagonist antibody refers to an agent that inhibits the activity of cytotoxic T lymphocyte-associated protein 4(CTLA-4) and thereby activates the immune system. CTLA-4 antagonists may bind to CTLA-4 and reverse CTLA-4-mediated immunosuppression. A non-limiting exemplary anti-CTLA-4 antibody is ipilimumab (BMS), which can be administered according to methods known in the art (e.g., methods approved by the FDA in the united states). For example, ipilimumab may be administered intravenously every three weeks in an amount of 3mg/kg over 90 minutes for a total of 4 doses (unresectable or metastatic melanoma); or administered intravenously in an amount of 10mg/kg every three weeks over 90 minutes for a total of 4 doses, then every 12 weeks in an amount of 10mg/kg for up to 3 years or until there is a recorded relapse or unacceptable toxicity (adjuvant melanoma).

OX40 agonist antibodies

In some embodiments, the one or more anti-cancer therapies are agonist anti-OX 40 antibodies. An OX40 agonist antibody is an agent that induces the activity of OX40, thereby activating the immune system and enhancing anti-tumor activity. Non-limiting exemplary agonist anti-OX 40 antibodies are Medi6469 (Medlmmone) and MOXR0916/RG7888 (Roche). These antibodies can be administered according to methods and protocols determined to be appropriate by those skilled in the art.

PD-1 therapy

In some embodiments, the one or more anti-cancer therapies are PD-1 therapies. PD-1 therapy encompasses any therapy that modulates the binding of PD-1 to PD-L1 and/or PD-L2. PD-1 therapy may, for example, interact directly with PD-1 and/or PD-L1. In some embodiments, the PD-1 therapy includes molecules that directly bind to and/or affect PD-1 activity. In some embodiments, PD-1 therapy includes molecules that directly bind to and/or affect the activity of PD-L1. Thus, antibodies that bind to PD-1 or PD-L1 and block the interaction of PD-1 with PD-L1 are PD-1 therapeutics. When the desired subtype of PD-1 therapy is intended, the phrase "PD-1 specific" will be used for therapies involving molecules that interact directly with PD-1, or the phrase "PD-L1 specific" will be used for molecules that interact directly with PD-L1, as the case may be. Unless otherwise specified, all disclosures herein relating to PD-1 therapy are generally applicable to PD-1 therapy, as well as PD-1-specific and/or PD-L1-specific therapies.

Non-limiting exemplary PD-1 therapies include nivolumab (A)BMS-936558, MDX-1106, ONO-4538); pidilizumab (pidilizumab), lambrolizumab (lambrolizumab)/pembrolizumab (KEYTRUDA, MK-3475); BGB-A317, tirelinzumab (tiselizumab) (BeiGene/Celgene); devolumab (durvalumab) (anti-PD-L1 antibody, MEDI-4736; AstraZeneca/Medlmmue); RG-7446; avelumab (avelumab) (anti-PD-L1 antibody; MSB-0010718C; Pfizer); AMP-224; BMS-936559 (anti-PD-L1 antibody); AMP-514; MDX-1105; a B-011; anti-LAG-3/PD-1; stevalizumab (CoStim/Novartis); anti-PD-1 antibody (Kadmon Pharm.); anti-PD-1 antibody (Immunovo); anti-TEVI-3/PD-l antibody (AnaptysBio); anti-PD-L1 antibody (CoStim/Novartis); RG7446/MPDL3280A (anti-PD-L1 antibody, Genentech/Roche); KD-033(Kadmon Pharm.); AGEN-2034 (Agenus); STI-A1010; STI-A1110; TSR-042; atelizumab (atezolizumab) (TECENTRIQ)^TM) (ii) a And other antibodies to programmed death protein 1(PD-1) or programmed death ligand 1 (PD-L1).

PD-1 therapy is administered according to protocols known in the art (e.g., U.S. FDA approved protocols). In one example, nivolumab is administered by intravenous infusion in an amount of 240mg every two weeks (unresectable or metastatic melanoma, adjuvant treatment of melanoma, non-small cell lung cancer (NSCLC), advanced renal cell carcinoma, locally advanced renal cell carcinoma, MSI-H or dMMR metastatic colorectal cancer and hepatocellular carcinoma) or 3mg/kg every three weeks (classical hodgkin lymphoma; recurrent or metastatic head and neck squamous cell carcinoma) over 60 minutes. In another example, pembrolizumab is administered by intravenous infusion in an amount of 200mg over 30 minutes once every three weeks. In another example, atelizumab is administered by intravenous infusion over 60 minutes in an amount of 1200mg every three weeks. In another example, avizumab is administered by intravenous infusion at 10mg/kg every two weeks over 60 minutes. In another example, Devolumab is administered by intravenous infusion in an amount of 10mg/kg over 60 minutes every two weeks.

TIGIT antagonists

In some embodiments, the one or more anti-cancer therapies is a TIGIT antagonist. TIGIT antagonists refer to agents capable of antagonizing or inhibiting the activity of T cell immune receptors (TIGIT) having Ig and ITIM domains, thereby reversing TIGIT-mediated immunosuppression. A non-limiting exemplary TIGIT antagonist is BMS-986207(Bristol-Myers Squibb/on Pharmaceuticals). These agents may be administered according to methods and protocols determined to be appropriate by those skilled in the art.

IDO inhibitors

In some embodiments, the one or more anti-cancer therapies are IDO inhibitors. IDO inhibitors refer to agents that are capable of inhibiting the activity of indoleamine 2, 3-dioxygenase (IDO) and thereby reversing IDO-mediated immunosuppression. IDO inhibitors may inhibit IDO1 and/or ID02(INDOL 1). The IDO inhibitor may be a reversible or irreversible IDO inhibitor. Reversible IDO inhibitors are compounds that reversibly inhibit IDO enzyme activity at either the catalytic or non-catalytic sites, while irreversible IDO inhibitors are compounds that irreversibly inhibit IDO enzyme activity by forming covalent bonds with the enzyme. Non-limiting exemplary IDO inhibitors are described in, for example, US 2016/0060237; and US 2015/0352206. Non-limiting exemplary IDO inhibitors include indoimod (Indoximod) (New Link Genetics), INCB024360(Incyte Corp), 1-methyl-D-tryptophan (New Link Genetics), and GDC-0919/navemomod (Genentech/New Link Genetics). These agents may be administered according to methods and protocols determined to be appropriate by those skilled in the art.

Ror gamma agonists

In some embodiments, the one or more anti-cancer therapies are ROR γ agonists. ROR γ agonists refer to agents that induce the activity of retinoic acid-related orphan receptor γ (ROR γ), thereby reducing immunosuppressive mechanisms. Non-limiting exemplary ROR gamma agonists include, but are not limited to, LYC-55716(Lycera/Celgene) and INV-71 (Innovammme). These agents may be administered according to methods and protocols determined to be appropriate by those skilled in the art.

b. Chemotherapy

In some embodiments, the one or more anti-cancer therapies are chemotherapeutic agents. Exemplary chemotherapeutic agents that may be used include, but are not limited to, capecitabine, cyclophosphamide, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine, irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, nabituarexate (nab-paclitaxel), ABRAXA(protein-bound paclitaxel), pemetrexed, vinorelbine, vincristine, erlotinib, afatinib, gefitinib, crizotinib, darafinib, trametinib, vemurafenib, and cobimetinib (cobimetanib). These agents may be administered according to methods and protocols determined to be appropriate by those skilled in the art.

c. Cancer vaccine

In some embodiments, the one or more anti-cancer therapies are cancer vaccines. Cancer vaccines have been investigated as potential methods for antigen transfer and activation of dendritic cells. In particular, vaccination in combination with immunological checkpoints or agonists against co-stimulatory pathways has shown evidence to overcome tolerance and generate increased anti-tumor responses. A range of cancer vaccines have been tested that employ different approaches to promote an immune response against a tumor (see, e.g., Emens LA, Expert Opin Emerg Drugs13(2):295-308 (2008)). Methods have been devised to enhance the response of B cells, T cells or professional antigen presenting cells to tumors. Exemplary types of cancer vaccines include, but are not limited to, those employing peptide-based vaccines targeting different tumor antigens, which may be delivered as peptides/proteins or as genetically engineered DNA vectors, viruses, bacteria, etc.; and cell biology approaches such as cancer vaccine development against less well-defined targets, including but not limited to vaccines developed from patient-derived dendritic cells, autologous tumor cells or tumor cell lysates, allogeneic tumor cells, and the like.

Exemplary cancer vaccines include, but are not limited to, dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, and the like. In some embodiments, such vaccines increase the anti-tumor response. Examples of cancer vaccines also include, but are not limited to, MAGE3 vaccine (e.g., for melanoma and bladder cancer), MUC1 vaccine (e.g., for breast cancer), EGFRv3 (such as Rindopepimut, e.g., for brain cancer, including glioblastoma multiforme), or ALVAC-CEA (e.g., for CEA + cancer).

Non-limiting exemplary cancer vaccines also include Sipuleucel-T derived from autologous Peripheral Blood Mononuclear Cells (PBMCs) including antigen presenting cells (see, e.g., Kantoff PW et al, N Engl J med.363:411-22 (2010)). In the Sipuleucel-T generation, PBMCs of patients are activated ex vivo by PA2024, a recombinant fusion protein of prostatic acid phosphatase (a prostate antigen) and granulocyte-macrophage colony stimulating factor (an immune cell activator). Another approach to candidate cancer vaccines is to generate an immune response against a particular peptide that is mutated in tumor tissue, such as melanoma (see, e.g., Carreno et al, Science 348:6236,2015). In some embodiments, such mutated peptides may be referred to as neoantigens (neoantigens). As a non-limiting example of the use of neoantigens in tumor vaccines, neoantigens in tumors that are expected to bind major histocompatibility complex protein HLA-a 02:01 have been identified for individual patients with cancer such as melanoma. Dendritic cells from a patient are matured ex vivo and then incubated with a neoantigen. The activated dendritic cells are then administered to the patient. In some embodiments, robust T cell immunity to neoantigens can be detected after administration of a cancer vaccine.

In some such embodiments, a cancer vaccine is developed using the neoantigen. In some embodiments, the cancer vaccine is a DNA vaccine. In some embodiments, the cancer vaccine is an engineered virus comprising a cancer antigen, such as PROSTVAC (rilimogen galvano/rilimogen glafolivec). In some embodiments, the cancer vaccine comprises engineered tumor cells, such as GVAX, which is a granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected tumor cell vaccine (see, e.g., Nemunaitis, Expert Rev. vaccines 4:259-274, 2005).

The vaccine may be administered according to methods and protocols determined to be appropriate by those skilled in the art.

d. Additional exemplary anti-cancer therapies

Additional non-limiting exemplary anti-cancer therapies include Luspatercept (Acceleron Pharma/Celgene); motolimod (Array BioPharma/Celgene/VentiRx Pharmaceuticals/Ligand); GI-6301 (GlobeIMmune/Celgene/NantWorks); GI-6200 (GlobeIMmune/Celgene/NantWorks); BLZ-945 (Celgene/Novartis); ARRY-382(Array BioPharma/Celgene), or any of the anti-cancer therapies provided in table 2. These agents may be administered according to methods and protocols determined to be appropriate by those skilled in the art. In some embodiments, the one or more anti-cancer therapies comprise surgery and/or radiation therapy. Thus, the anti-cancer therapy may optionally be used in an adjuvant or neoadjuvant setting.

e. Combination of

In various embodiments, the anti-cancer therapy administered to the patient is a combination of one or more (e.g., two, three, or more) anti-cancer therapies including, for example, one or more of the anti-cancer therapies listed above or elsewhere herein.

In various examples, an anti-ICOS agonist antibody (e.g., an antibody described above, such as JTX-2011) is administered in combination with another immunotherapy (see, e.g., above). In one example, an anti-ICOS agonist antibody (e.g., an antibody described above, such as JTX-2011) is administered in combination with a PD-1 therapy (e.g., a PD-1 therapy listed above). Thus, in various examples, the invention comprises administering an anti-ICOS agonist antibody (e.g., JTX-2011) in combination with one or more of: nivolumab, pidilizumab, lanborrelizumab/pembrolizumab, BGB-A317, tirezuzumab, Devolumab, RG-7446, Avermezumab, AMP-224, BMS-936559, AMP-514, MDX-1105, A B-011, anti-LAG-3/PD-1, and sibutrumab (Costim/Novartis); anti-PD-1 antibody (Kadmon Pharm.); anti-PD-1 antibody (Immunovo); anti-TEVI-3/PD-l antibody (AnaptysBio); anti-PD-L1 antibody (CoStim/Novartis); RG7446/MPDL3280A, KD-033(Kadmon Pharm.); AGEN-2034(Agenus), STI-A1010, STI-A1110, TSR-042, atlas, and other antibodies directed against programmed death protein 1(PD-1) or programmed death ligand 1 (PD-L1). In one specific example, JTX-2011 is administered with nivolumab.

Optionally, the above combination further comprises one or more additional anti-cancer agents (e.g., immunotherapy). Thus, the above combinations may optionally include one or more of the following: anti-CTLA-4 antagonist antibodies (e.g., ipilimumab), anti-OX 40 antibodies (e.g., Medi6469 or MOXR0916/RG7888), TIGIT antagonists (e.g., BMS-986207), IDO inhibitors (e.g., indoimod, INCB024360, 1-methyl-D-tryptophan, or GDC-0919/natavamod), ROR γ agonists (e.g., LYC-55716 and INV-71), or chemotherapeutic agents (see e.g., above) or cancer vaccines (see e.g., above).

In other examples, a combination of the invention includes an anti-ICOS agonist antibody (e.g., an antibody described above, such as JTX-2011) and one or more of: anti-CTLA-4 antagonist antibodies (e.g., ipilimumab), anti-OX 40 antibodies (e.g., Medi6469 or MOXR0916/RG7888), TIGIT antagonists (e.g., BMS-986207), IDO inhibitors (e.g., indoimod, INCB024360, 1-methyl-D-tryptophan, or GDC-0919/natavamod), ROR γ agonists (e.g., LYC-55716 and INV-71), or chemotherapeutic agents (see e.g., above) or cancer vaccines (see e.g., above).

In various examples, the components of the combination are administered according to a dosing regimen described herein (e.g., a U.S. FDA-approved dosing regimen; see above) or using other regimens determined to be appropriate by one of skill in the art.

Pharmaceutical compositions and administration

Compositions comprising one or more anticancer therapies are provided in formulations having a wide variety of pharmaceutically acceptable carriers, as determined to be appropriate by those skilled in The art (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with products and Comparisons: drugs Plus, 20 th edition (2003); Ansel et al, Pharmaceutical food Forms and Drug Delivery Systems, 7 th edition, Lippincott, Williams and Wilkins (2004); Kibbe et al, Handbook of Pharmaceutical Excipients, 3 rd edition, Pharmaceutical Press (2000). various pharmaceutically acceptable carriers (including vehicles, adjuvants and diluents) are useful Dextrose, water, glycerol, ethanol, and combinations thereof.

Anti-cancer therapies are administered in the practice of the methods of the invention as known in the art (e.g., according to FDA approved protocols) or as indicated elsewhere herein (see, e.g., above). In some embodiments, the anti-cancer therapy of the present invention is administered in an amount effective to treat cancer. A therapeutically effective amount will generally depend upon the weight of the subject being treated, his or her physical or health status, the breadth of the condition being treated, the age of the subject being treated, the method of pharmaceutical formulation, and/or the method of administration (e.g., time of administration and route of administration).

In some embodiments, the anti-cancer therapy may be administered in vivo by various routes including, but not limited to, intravenous, intra-arterial, parenteral, intratumoral, intraperitoneal, or subcutaneous. One skilled in the art can select the appropriate formulation and route of administration depending on the intended application.

Exemplary methods for detecting Total ICOS and/or T-beta expression levels

Provided herein are methods of assessing a patient's responsiveness to one or more anti-cancer therapies. In some embodiments, methods are provided for identifying subjects who may benefit from continued treatment with one or more anti-cancer therapies, optionally in combination with an anti-ICOS agonist antibody.

a. Exemplary antibody-based detection methods

In some embodiments, the methods comprise using, for example, an anti-ICOS and/or anti-T-beta antibody, polypeptide, or polynucleotide to determine whether a patient treated with one or more anti-cancer therapies has CD4+ T cells with increased ICOS and/or T-beta expression in peripheral blood. In some embodiments, the detection method comprises contacting a patient sample (e.g., a peripheral blood sample or a portion thereof) with an antibody, polypeptide, or polynucleotide and determining whether the level of binding is different from the level of binding of a control. In some embodiments, CD4+ T cells from a peripheral blood test sample are contacted with an anti-ICOS detection antibody and/or an anti-T-beta detection antibody, and binding between the antibody(s) and CD4+ T cells is determined. When an increase in binding activity of CD4+ T cells from the test sample to the antibody(s) is shown compared to CD4+ T cells from the control sample, continued treatment with one or more anti-cancer therapies, optionally in combination with anti-ICOS agonist antibody treatment, as described herein, is indicated.

Various methods known in the art for detecting specific antibody-antigen binding can be used. Such assays include, but are not limited to, flow cytometry (including, for example, Fluorescence Activated Cell Sorting (FACS)), indirect immunofluorescence, solid phase enzyme-linked immunosorbent assay (ELISA), ELISpot assays, Fluorescence Polarization Immunoassay (FPIA), Fluorescence Immunoassay (FIA), Enzyme Immunoassay (EIA), turbidity inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and Radioimmunoassay (RIA), Western blotting (including cellular Western), immunofluorescent staining, micro-engraving (micrograving) (see Han et al,lab Chip 10(11):1391-1400,2010), Quant-iT and Qubit protein assay kits, NanoOrange protein quantification kits, CBQCA protein quantification kits, EZQ protein quantification kits, Click-iT reagents, Pro-Q Diamond phosphoprotein staining, Pro-Q glycoprotein staining kits, peptide and protein sequencing, N-terminal amino acid analysis (Life science Technologies, Grand Island, NY), chemiluminescence or colorimetric-based ELISA cytokine arrays (Signosis), Intracellular Cytokine Staining (ICS), BD Photoflow^TMAnd BD^TMCytometric bead arrays (BD Sciences, San Jose, CA); CyTOF mass cytometry (DVS Sciences, Sunnyvale CA); mass spectrometry, microplate capture and detection assays (Thermo Scientific, Rockland, IL), multiplexing techniques (e.g. Luminex, Austin, TX); FlowCellect^TMT cell activation kit (EMD Millipore); surface Plasmon Resonance (SPR) -based techniques (e.g., Biacore, GE Healthcare Life Sciences, Uppsala, Sweden); CD4+ effector memory T cell isolation kits and CD8+ CD45RA + effector T cell isolation kits (Miltenyi Biotec inc., CA); EasySep^TMHuman T cell enrichment kit (StemCells, inc., Vancouver, Canada); human Thl/Th2/Thl7 phenotyping kit (BD Biosciences, CA); immunofluorescent staining of incorporated bromodeoxyuridine (BrdU) or 7-aminoactinomycin D. See also Current Protocols in Immunology (2004) section 3.12.1-3.12.20 publishers: john Wiley&Sons, inc, or Current Protocols in Immunology (2013) publishers: john Wiley&Sons, inc, the contents of which are incorporated herein by reference in their entirety.

Indicator moieties or labeling groups can be attached to the subject antibodies and selected to meet the needs of various uses of the method, typically dictated by the availability of the assay device and compatible immunoassay procedures.

Suitable labels include but are not limited to radionuclides (e.g.,¹²⁵I、¹³¹I、³⁵S、³h or³²P), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, luciferase or beta-galactosidase), a fluorescent moiety or protein (e.g., fluorescein, rhodamine, phycoerythrin, GFP or BFP), or a luminescent moietyAnd (e.g., Qdot supplied by Quantum Dot Corporation, Palo Alto, Calif.)^TMNanoparticles). General techniques for performing the various immunoassays described above are known to those of ordinary skill in the art.

In some cases, the anti-ICOS detection antibody need not be labeled, and a second labeled antibody that binds to the first anti-ICOS antibody can be used to detect its presence.

In some cases, the anti-T-beta detection antibody need not be labeled, and a second labeled antibody that binds to the first anti-T-beta antibody can be used to detect its presence.

In some embodiments, CD4+ cells from a peripheral blood test sample are contacted with an anti-ICOS detection antibody and/or an anti-T-beta detection antibody, and binding between the antibody(s) and CD4+ cells is determined. In some embodiments, the expression level of total ICOS and/or T-beta in CD4+ T cells is determined using a fluorescence activated cell sorter. Fluorescence activated cell sorters may have varying degrees of complexity, such as multiple color channels, low and obtuse angle light scatter detection channels, impedance channels, and the like. Cells can be selected for dead cells by using dyes associated with dead cells (e.g., propidium iodide). FACS apparatus typically include a light source (typically a laser) and several detectors for detecting cell particles or cell subsets in a mixture using light scattering or light emission parameters. The basic mechanism of FACS is well known in the art and basically involves scanning (e.g., counting, sizing or fluorescent label sorting) individual particles flowing through a laser source in a liquid medium. When light from an excitation source strikes a moving particle, the light is scattered and fluoresces. Forward scattering (FSC, light scattered in the forward direction (i.e., the same direction as the light beam)) provides basic morphological information about the particle, such as cell size and morphology. Light scattered at 90 ° from the incident beam is due to refraction or reflection of light and is called side angle scattering (SSC). This parameter measures particle size and cell surface topology of the particles. In summary, the scattered signals in the forward and wide angle directions are used to identify cell subsets based on cell size, morphology and granularity. This information is used to distinguish various cell populations in heterogeneous samples.

Exemplary anti-ICOS antibodies for use in the detection aspects of the methods described herein are antibodies that recognize an internal (i.e., intracellular) epitope of ICOS. Although ICOS may be expressed on the surface of T cells, it is estimated that a significant proportion of total cellular ICOS is present in the intracellular stores (e.g., about 80%). While exemplary therapeutic anti-ICOS antibodies (such as JTX-2011) recognize extracellular epitopes of ICOS, the use of anti-ICOS detection antibodies that specifically bind intracellular ICOS epitopes allows for determination of total ICOS expression levels. Examples of antibodies that recognize intracellular ICOS epitopes and thus can be used in methods for detecting total ICOS include 2M13 and 2M19 (see WO 2017/070423; see also table 3 below) and variants thereof. In addition, antibodies that compete with 2M13 and 2M19 for binding to ICOS can be used to detect ICOS according to the methods of the invention.

b. Exemplary nucleic acid-based detection methods

In some embodiments, the methods provided herein comprise measuring mRNA levels. In some embodiments, the methods provided herein comprise measuring ICOS and/or T-beta mRNA.

Any suitable method of determining mRNA levels may be used. Methods for evaluating mRNA include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the target sequence, Northern blotting, and related techniques) and various nucleic acid amplification assays (such as RT-PCR and other amplification type detection methods using complementary primers specific for the target sequence, e.g., branched DNA, SISB a, TMA, and the like).

In some embodiments, mRNA levels are determined by quantitative RT-PCR. In some embodiments, mRNA levels are determined by digital PCR. In some embodiments, mRNA levels are determined by RNA-Seq. In some embodiments, mRNA levels are determined by an Rnase Protection Assay (RPA). In some embodiments, mRNA levels are determined by Northern blotting. In some embodiments, mRNA levels are determined by In Situ Hybridization (ISH). In some embodiments, the mRNA level is determined by a method selected from the group consisting of quantitative RT-PCR, microarray, digital PCR, RNA-Seq, Rnase Protection Assay (RPA), Northern blot, and In Situ Hybridization (ISH).

In some embodiments, e.g., when using quantitative RT-PCR, the threshold cycle numbers between two mrnas are compared, and a lower threshold indicates a higher level of the corresponding mRNA. As a non-limiting example, in some embodiments, if the levels of ICOS mRNA and at least one reference mRNA are analyzed and the threshold cycle number (Ct) of ICOS is 28 and the Ct of the reference mRNA is 30, ICOS has a higher level than the reference. In various embodiments, similar comparisons can be made for any type of quantitative or semi-quantitative analytical method.

In some embodiments, the level of at least one mRNA is normalized. In some embodiments, the levels of at least two mrnas are normalized and compared to each other. In some embodiments, such normalization may allow for comparison of mRNA levels when levels cannot be determined simultaneously and/or in the same assay reaction. One skilled in the art can select a suitable normalization basis, such as at least one reference mRNA or other factor, depending on the assay.

In some embodiments, the at least one reference mRNA comprises a housekeeping gene. In some embodiments, the at least one reference mRNA comprises one or more of RPLP0, PPIA, TUBB, ACTB, YMHAZ, B2M, UBC, TBP, GUSB, HPRT1, or GAPDH.

VI. examples

The following discussed embodiments are intended only as examples of the present invention and should not be construed as limiting the invention in any way. The examples are not intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Example 1: examination of Total ICOS expression in CD4+ T cells of cancer patients receiving JTX-2011 monotherapy or JTX-2011 and Natuzumab combination therapy

Design of research

Total ICOS expression in CD4+ T cells of 44 patients with gastric cancer, non-small cell lung cancer (NSCLC) or Triple Negative Breast Cancer (TNBC) who received JTX-2011 monotherapy (0.3mg/kg q3w) or JTX-2011(0.1mg/kg or 0.3mg/kg q3w) and nivolumab (240mg q3w) in combination therapy was evaluated using multi-color flow cytometry as described below. Of these patients, 4 had a confirmed partial response (cPR), 3 had an unconfirmed Partial Response (PR), 17 had Stable Disease (SD), and 20 had Progressive Disease (PD) as the Best Overall Response (BOR) in response to therapy.

Assessment of total ICOS expression in CD4+ T cells by flow cytometry

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from patient whole blood samples by density gradient separation using a BD Vacutainer CPT mononuclear cell preparation. The isolated PBMC samples were then frozen and stored at-80 ℃ until used in flow cytometry applications. At the time of analysis, the PBMC sample tubes were thawed in a 37 ℃ water bath for approximately 2 minutes. Each sample was then transferred to a 15mL conical tube with FACS buffer (1x PBS, 2% FBS, 0.01% sodium azide, 2mM EDTA) and cells were counted. Each sample pair 1x10⁶Individual PBMCs were stained. After counting, PBMCs were centrifuged at 500xg for 3 minutes to obtain cell pellets. Excess buffer was aspirated and the cell pellet was resuspended in FACS buffer. The cell pellet resuspension was evenly divided into wells of a 96-well round bottom plate, and then every 1 × 10⁵Each well was Fc-blocked for 20 min at room temperature using 5 μ L of Fc block (Human TruStain FcX, BioLegend catalog No. 422302). After blocking, the plates were centrifuged at 500Xg for 3 minutes and excess buffer was removed.

Wells designated as preliminary staining mixes to assess total ICOS levels received 100 μ L of a master staining mix containing anti-human CD3 (clone: UCHT1), anti-human CD4 (clone: OKT4), and JTX-2011 Dylight 650. Wells designated as isotype staining mixtures received 100 μ L of master staining mixture containing anti-human CD-3 (clone: UCHT1), anti-human CD4 (clone: OKT4), and anti-RSV Dylight 650. The staining mixture was incubated at 4 ℃ for 30 minutes and then centrifuged at 500Xg for 3 minutes, washed twice with FACS buffer. All wells were then fixed and permeabilized for 30 minutes (eBioscience FOXP3// transcription factor staining buffer kit, ref #00-5523-00 Life Technologies). After permeabilization, plates were centrifuged at 500Xg for 3 minutes and excess buffer was removed. Wells designated as preliminary staining mixtures received 100 μ L of a master staining mixture diluted in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, reference #00-5523-00 Life Technologies) containing anti-T-beta (clone: 4B10), streptavidin PE (BioLegend catalog 405204) and an internal epitope recognizing ICOS (see fig. 1) was biotinylated M13 anti-ICOS detection antibody (joint Therapeutics).

Wells designated as isotype control staining mixtures received 100 μ L of the master staining mixture containing only streptavidin PE prepared in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, reference #00-5523-00 Life Technologies).

The staining mixture was incubated at 4 ℃ for 30 minutes. The plate was then centrifuged at 500Xg for 3 minutes and washed twice with 1 Xpermeabilization buffer. The contents of the wells were then resuspended in 150 μ L FACS buffer. Stained samples were immediately analyzed using a BD FACS Canto flow cytometer and the resulting data analyzed using FlowJo analysis software.

FIG. 2 illustrates determining whether a sample includes ICOS^hiAnalysis of the CD4+ T cell population of (1). Dragging gating for partitioning into ICOS^loPopulation and ICOS^hiPopulation and coverage of ICOS with calculated geometric mean fluorescence intensity^loAnd ICOS^hiA histogram of the quadrant.

Results

ICOS was observed in gastric cancer patients with cPR for JTX-2011(0.3mg/kg, q3w) monotherapy^hiThe appearance and stabilization of the CD4+ T cell population. The population was detected as early as after cycle 3, expanded and stabilized after cycle 15 (fig. 3A). ICOS^hiThe appearance and stability of the CD4+ T population correlated with evidence of clinical activity, as evidenced by a reduction in target lesion size as assessed according to RECIST 1.1 criteria (fig. 3B).

In pairs of JTX-2011(0.1mg/kg, q3w) and NaCombination therapy of Wumab (240mg, q3w) an elevated ICOS expression level (ICOS) was also observed in samples from gastric cancer patients with cPR^hi) The CD4+ T cell population of (1). This population was detected after cycle 7 and had subsequent population stabilization after passage through at least cycle 11 (fig. 4).

ICOS was observed in samples from gastric cancer patients who presented stable disease in response to combination therapy of JTX-2011(0.3mg/kg, q3w) and nivolumab (240mg, q3w)^hiTransient population of CD4+ T cells (fig. 5). ICOS before disease progression^hiThe CD4+ T cell population was observed after cycle 4 and expanded after cycle 5, but decreased after cycle 6.

ICOS was not observed in TNBC patients exhibiting SD or PD in response to combination therapy of JTX-2011(0.3mg/kg, q3w) and nivolumab (240mg, q3w)^hiCD4+ T cell population (fig. 6).

Conclusion

ICOS was observed in all of the cPR and PR patients and in 11 of 17 patients with stable disease as a therapy-responsive BOR^hiCD4+ T cell population. This population was not observed in the remaining 6 patients with stable disease, nor in the 20 patients with progressive disease in response to therapy (fig. 7A-7C). The appearance of this population correlated with evidence of biological activity corresponding to the baseline percentage change in target lesion size (fig. 7A-7C).

Example 2: examination of Total ICOS expression in CD4+ T cells of Sa1/N tumor-bearing mice receiving JTX-1011-mG2a

Design of research

Mice bearing Sa1/N fibrosarcoma (Ostrand-Rosenberg,2001, Curr. Protoc. Immunol., chapter 20) received a weekly dose of 0.25mG/kg JTX-1011-mG2 a. Whole blood samples were collected 1 hour and 48 hours after administration of the second dose of antibody and analyzed for total ICOS expression in CD4+ T cells as described below.

Assessment of total ICOS expression in CD4+ T cells by flow cytometry

Peripheral blood samples were collected via the tail vein into BD NaEDTA Microtainer tubes and freshly stained for flow cytometry analysis. 100 μ L of whole blood was dispensed into appropriate wells in a 96-well round bottom plate, and then each well was Fc-blocked for 15 minutes at 4 ℃. Fc blocking was performed using TruStain fcX (anti-mouse CD16/32) antibody (BioLegend, Cat. 101320).

Wells designated as primary staining mixes to assess total ICOS levels received 100 μ L of the master staining mix containing anti-CD 3 (clone 145-2C11), anti-CD 4 (clone GK1.5), anti-CD 8 (clone 53-6.7), anti-ICOS (JTX-2011 DyLight650, joint Therapeutics). Wells designated as isotype staining mixtures received 100 μ L of master staining mixture containing species and fluorophore specific isotype controls. The staining mixture was incubated at 4 ℃ for 30 minutes and then centrifuged at 500Xg for 3 minutes, washed twice with FACS buffer. All wells were then fixed and permeabilized for 30 minutes (eBioscience FOXP3// transcription factor staining buffer kit, ref #00-5523-00 Life Technologies). After permeabilization, plates were centrifuged at 500Xg for 3min and excess buffer was removed. Wells designated as preliminary staining mixtures received 100 μ L of the master staining mixture containing FOXP3 (clone FJK-16s) prepared in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, reference #00-5523-00 Life Technologies).

Wells designated as isotype control staining mixtures received 100 μ L of the master staining mixture containing rat IgG2a, kappa isotype control antibodies prepared in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, ref #00-5523-00 Life Technologies).

The staining mixture was incubated at 4 ℃ for 30 minutes. The plate was then centrifuged at 500Xg for 3 minutes and washed twice with 1 Xpermeabilization buffer. The wells were then resuspended in 150 μ L FACS buffer. Stained samples were immediately analyzed on a BD FACS Canto flow cytometer and the resulting data analyzed using FlowJo analysis software.

Results and conclusions

An increase in ICOS staining was observed 48 hours post-dose for the second cycle of JTX-1011-mG2a relative to 1 hour post-dose (FIG. 8A). The increase in staining corresponded to a distinct ICOS at 48 hours after JTX-1011-mG2a administration^hiRapid emergence of CD4+ T cell population (fig. 8B).

Example 3: examination of Total ICOS and T-beta expression in CD4+ T cells of cancer patients receiving JTX-2011 monotherapy or combination therapy of JTX-2011 and Natuzumab

Design of research

Total ICOS and T-bet expression in CD4+ T cells of patients with gastric, Triple Negative Breast (TNBC), or endometrial cancer receiving JTX-2011 monotherapy (0.3mg/kg q3w) or JTX-2011(0.1mg/kg or 0.3mg/kg q3w) and nivolumab (240mg q3w) in combination therapy is evaluated using multi-color flow cytometry as described below.

Assessment of Total ICOS and T-beta expression in CD4+ T cells by flow cytometry

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from patient whole blood samples by density gradient separation using a BD Vacutainer CPT mononuclear cell preparation. At the time of analysis, the PBMC sample tubes were thawed in a 37 ℃ water bath for approximately 2 minutes. Each sample was then transferred to a 15mL conical tube with FACS buffer (1x PBS, 2% FBS, 0.01% sodium azide, 2mM EDTA) and cells were counted. Each sample pair 1x10⁶Individual PBMCs were stained. After counting, PBMCs were centrifuged at 500xg for 3 minutes to obtain cell pellets. Excess buffer was aspirated and the cell pellet was resuspended in FACS buffer. The cell pellet resuspension was evenly divided into wells of a 96-well round bottom plate, and then every 1 × 10⁵Each well was Fc-blocked for 20 min at room temperature using 5 μ L of Fc block (Human TruStain FcX, BioLegend catalog No. 422302). After blocking, the plates were centrifuged at 500Xg for 3 minutes and excess buffer was removed.

Wells designated as preliminary staining mixes to assess total ICOS levels received 100 μ L of a master staining mix containing anti-human CD3 (clone: UCHT1), anti-human CD4 (clone: OKT4), and JTX-2011 Dylight 650. Wells designated as isotype staining mixtures received 100 μ L of master staining mixture containing anti-human CD-3 (clone: UCHT1), anti-human CD4 (clone: OKT4), and anti-RSV Dylight 650. The staining mixture was incubated at 4 ℃ for 30 minutes and then centrifuged at 500Xg for 3 minutes, washed twice with FACS buffer. All wells were then fixed and permeabilized for 30 minutes (eBioscience FOXP3// transcription factor staining buffer kit, ref #00-5523-00 Life Technologies). After permeabilization, plates were centrifuged at 500Xg for 3 minutes and excess buffer was removed. The wells designated as the preliminary staining mixture received 100 μ L of a master staining mixture diluted in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, reference #00-5523-00 Life Technologies) containing anti-T-beta (clone: 4B10), streptavidin PE and an internal epitope that recognizes ICOS (see fig. 1) is biotinylated M13 anti-ICOS detection antibody.

Wells designated as isotype control staining mixtures received 100 μ L of the master staining mixture containing only streptavidin PE prepared in 1x permeabilization buffer (eBioscience FOXP 3/transcription factor staining buffer kit, reference #00-5523-00 Life Technologies).

The staining mixture was incubated at 4 ℃ for 30 minutes. The plate was then centrifuged at 500Xg for 3 minutes and washed twice with 1 Xpermeabilization buffer. The contents of the wells were then resuspended in 150 μ L FACS buffer. Stained samples were immediately analyzed using a BD FACS Canto flow cytometer and the resulting data analyzed using FlowJo analysis software.

Results and conclusions

ICOS was observed in PBMC samples from gastric cancer patients with cPR for combination therapy of JTX-2011(0.1mg/kg, q3w) and nivolumab (240mg, q3w)^hiThe appearance and stabilization of the CD4+ T cell population. This population was first detected at cycle 10 and further identified as containing a T-beta level (T-beta)^hi) Elevated ICOS^hiCD4+ T cell subset (fig. 9).

ICOS was also observed in samples from gastric cancer patients with cPR monotherapy for JTX-2011(0.3mg/kg, q3w)^hi/T-bet^hiEmergence of CD4+ T cell population. This population was detected after cycle 3 and had subsequent population stabilization after passage of at least cycle 15 (fig. 10).

ICOS was not observed in TNBC and endometrial cancer patients exhibiting SD in response to combination therapy of JTX-2011(0.3mg/kg, q3w) and nivolumab (240mg, q3w)^hi/T-bet^hiCD4+ cellsClusters (fig. 11 and 12).

Example 4: ICOS^hiExpansion of CD4+ T cell population

4.1

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with OKT-3+ IL-2 to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with nivolumab (anti-PD-1) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.2

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with OKT-3+ IL-2+ ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with nivolumab (anti-PD-1) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.3

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (Cat. No. 10971) to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with nivolumab (anti-PD-1) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.4

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (cat. No. 10971) + ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with nivolumab (anti-PD-1) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.5

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with OKT-3+ IL-2 to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with ipilimumab (anti-CTLA-4) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.6

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells 500uL dispensed into wells of 48-well plates supplemented with 5% human AB serum (Sigma H4522) and 1%Antibiotic antifungal solution (Sigma A5955) in AIM-V medium (Thermo A3830801).

The medium was further supplemented with OKT-3+ IL-2+ ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with ipilimumab (anti-CTLA-4) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.7

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (Cat. No. 10971) to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with ipilimumab (anti-CTLA-4) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.8

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (cat. No. 10971) + ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation. The medium was further supplemented with ipilimumab (anti-CTLA-4) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.9

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with OKT-3+ IL-2 to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation (except OKT-3) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance, including OKT-3. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.10

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with OKT-3+ IL-2+ ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation (except OKT-3) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance, including OKT-3. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.11

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (Sigma A5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (Cat. No. 10971) to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation (except Stemcell ImmunoCult human CD3/CD 28T cell activator) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form.

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance, including Stemcell ImmunoCult human CD3/CD 28T cell activator. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

4.12

PBMCs from healthy donors were dosed at approximately 3x10 per well⁵Density of individual cells was distributed to 500uL of AIM-V medium (Thermo A3830801) supplemented with 5% human AB serum (Sigma H4522) and 1% antibiotic antifungal solution (SigmaA5955) in wells of 48-well plates.

The medium was further supplemented with IL-2+ IL-12+ anti-IL-4 + Stemcell ImmunoCult human CD3/CD 28T cell activator (cat. No. 10971) + ICOS-L to induce ICOS expression in CD4+ T cells. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for 3 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation (except Stemcell ImmunoCult human CD3/CD 28T cell activator) to maintain ICOS expression in CD4+ T cells and prevent depletion. All supplements were delivered in soluble form, except ICOS-L, which had previously been coated onto culture plates (plate-bound form).

Cells were incubated at 37 ℃ for an additional 4 days and then transferred to new wells containing fresh medium and all supplements for initial stimulation and ICOS maintenance, including Stemcell ImmunoCult human CD3/CD 28T cell activator. The cells were then incubated at 37 ℃ for an additional 3 days, then stained and fixed. At the end of a total of 10 days of incubation, cells were expanded a minimum of 24-fold.

Example 5: evaluation of antigen-specific ICOS^hiAnd ICOS^loCytokine response in CD4+ T cells

Design of research

Stimulation of PBMC from healthy donors to induce ICOS using tetanus toxoid as a model recall antigen^hiCD4+ T cell population. After 24 hours of stimulation with antigen, cells were washed to remove the stimulus and left CD4+ T cells. After washing, soluble JTX-2011 was added and intracellular cytokine production was assessed by flow cytometry after 6 hours of incubation in the presence of brefeldin a.

Results and conclusions

Only if there is an ICOS already present^hiEx vivo stimulation of soluble JTX-2011 was only effective with CD4+ T cells. JTX-2011 induces an effective multifunctional cytokine response characterized by antigen-specific ICOS^hiMid rather than ICOS^loBoth IFN γ and TNF α increased on average 4-fold in CD4+ T cells (fig. 13).

Example 6: evaluation of PD-1 inhibition vs ICOS^hiEffect of CD4+ T cell appearance

Design of research

Samples of subjects receiving standard-of-care PD-1 inhibitor treatment were obtained from commercial biological repositories. Overall, the appearance of a CD4+ T cell population from PBMCs of 77 subjects (mainly those with lung cancer or melanoma) was assessed by flow cytometry profiling (fig. 14A).

Results and conclusions

Longitudinal flow spectra of NSCLC subjects responsive to nivolumab and NSCLC subjects responsive to pembrolizumab did not show evidence of ICOS^hiInduction of CD4+ T cells (fig. 14B). Thus, ICOS^hiThe appearance of the CD4+ T cell population was associated with JTX-2011 activity, but not with PD-1 inhibition.

Example 7: ICOS^hiAnd ICOS^loTranscriptional and phenotypic profiling of CD4+ T cells

Design of research

Therapeutic ICOS development Using Nanostring human immunological Panel (human immunology)^hiPurified CD4+ T cells from the subject of cells were subjected to transcriptional analysis with CD4+ T cells from a reference cancer patient who did not show a population of cells. In addition, the immunophenotype of peripheral blood T cells was assessed by flow cytometry at various time points before and after treatment with JTX-2011 alone or in combination with nivolumab.

Results and conclusions

ICOS as demonstrated by both transcriptional profiling and immunophenotypic evaluation by flow cytometry^hiCD4+ T cells were found to interact with ICOS^loThe cells are different. When unsupervised clustering was performed using Pearson correlation coefficients, different clusters were formed for patient and donor CD4+ T cell samples (fig. 15A). Gene set enrichment analysis demonstrated trends in modulating several pathways (FIG. 15B), and ICOS in particular^hiCD4+ T cells were found to be rich in effector pathways. In particular, with ICOS^loCD4+ T cells compared, allograft rejection pathway components (depicted in FIG. 15C) were found in ICOS^hiUp-regulated in CD4+ T cells.

Uniform ICOS at later cycles^hiEvaluation of lineage and activation markers in subjects of the population revealed, ICOS^hiCD4+ T cells were predominantly T effector cells of the Th1 lineage (fig. 16A). ICOS^hiCD4+ T cells are not rich in tregs. Baseline and in-treatment analysis of gastric cancer subjects with cPR using the T-distribution stochastic neighbor embedding (tSNE) clustering algorithm demonstrated an overall decrease in LAG-3 expression and an increase in TIGIT expression on non-Treg cells (FoxP3-) after JTX-2011 treatment (fig. 16B). In general, ICOS is emerging^hiIncreased activation, but not depletion, of Tbet + non-Treg CD4+ and CD8+ T cells was observed in the subjects of the population.

Longitudinal analysis of mean Ki-67 staining in subjects with confirmed PR after JTX-2011 treatment demonstrated that CD8+ and CD4+ T cells had early and late proliferation, respectively, characterized by the appearance of ICOS^hiBiphasic proliferation in subjects of the population (fig. 17).

Example 8: examination of the clonality of the T cell receptor Bank after treatment with JTX-2011

Design of research

The clonality of the T cell receptor pool evaluated on peripheral T cells and archived tumor tissue was determined using Adaptive biotechnology ImmunoSeq.

Results and conclusions

Analysis of changes in clonal abundance in the peripheral blood T Cell Receptor (TCR) pool was expanded in significant treatment of clones identified in 18/22 (about 82%) subjects (including those of monotherapy) after JTX-2011 treatment. The longitudinal profile of the JTX-2011-induced peripheral clonality changes demonstrated biphasic amplification of circulating pools (fig. 18). Longitudinal profiles of bystander and tumor associated clones demonstrated in the absence of ICOS^hiThere was no distinct polyclonal expansion of TCR clones in representative subjects with CD4+ T cell presentation (fig. 19A). In the presence of ICOS^hiClonal expansion was also observed in subjects with CD4+ T cells, but greater expansion of tumor-associated clones relative to bystander (fig. 19B). The amplified clones detected in the periphery were tumor-associated clones present in archived tumor samples, suggesting that JTX-2011 may play a role in enhancing cell-mediated anti-tumor immunity.

Overall, TCR clonality evaluation demonstrated clonal expansion in treatment and showed ICOS^hiTumor-associated clones have greater expansion in subjects with the CD4+ T cell phenotype, whereas JTX-2011 treatment results in expansion of de novo T cell clones that are associated with ICOS^hiCD4+ T cells appeared irrelevant.

Example 9: analysis of PBMC from responding patients

Design of research

Selection from a plant with confirmed PR with homogeneous ICOS^hiPBMCs of subjects of the CD4+ T cell population to assess antigen specificity. PBMC stimulation was performed and IFN γ secretion was detected using an ELISPOT reader. Peptides were tested as pools containing 2. mu.g/mL of each mutant peptide.

Results and conclusions

Flow cytometry analysis demonstrated ICOS at selected time points^hiThe CD4+ T cell population was homogeneous (fig. 20A). Analysis of PBMCs from responding patients showed that this was observed during treatmentTumor(s)Antigen-specific immune responses (fig. 20B).

Example 10: checking ICOS^hiOccurrence and survival of

Design of research

PBMCs from a 50 patient subgroup with an ICONIC study of evaluable samples were phenotyped by flow cytometry over time. Clinical features and results were analyzed, including uncorrected p-values for post hoc statistical analysis.

Results and conclusions

The appearance of overt and persistent ICOS^hiThe peripheral CD4+ T cell population was associated with improved survival following JTX-2011 monotherapy and combination therapy with nivolumab (figure 21) (with ICOS)^hiThe median of CD4+ T patients was 6.2 months, with ICOS alone^loCD4+ T cell patients and all patients in the study (including unanalyzed ICOS)^hiThose patients with T cell appearance) for 2 months of comparison). The apparent ICOS appeared^hiThe peripheral CD4+ T cell population was also associated with OS improvement (fig. 22) (with ICOS)^hiThe median number of patients with CD4+ T cells has not yet been reached, compared to ICOS alone^loPatients with CD4+ T cells were 9 months compared to 9.3 months for all patients with the ICONIC study).

The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

TABLE 2

TABLE 3 sequences