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1. (WO2019032927) METHODS FOR PRODUCING GENETICALLY ENGINEERED CELL COMPOSITIONS AND RELATED COMPOSITIONS
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CLAIMS

WHAT IS CLAIMED:

1. A method for generating a cell composition, the method comprising:

combining a first cell composition comprising naive-like CD4+ T cells with a second cell composition comprising naive-like CD8+ T cells to produce an input cell composition in which the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is between or about between 0.8: 1 and 2.2: 1, inclusive.

2. The method of claim 1, wherein the first cell composition is produced by isolating CD4+ T cells from a biological sample obtained from a subject and/or the second cell composition is produced by isolating CD8+ T cells from the biological sample obtained from the subject.

3. The method of claim 1 or claim 2, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the naive-like CD4+ T cells in the first cell composition and/or the number, number per volume, number per weight, and/or percentage of the naive-like CD8+ T cells in the second cell composition.

4. The method of claim 2, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the naive-like CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of naive-like CD8+ T cells in the biological sample from the subject.

5. The method of any of claims 1-4, wherein the ratio of the naive-like CD4+ T cells to naive-like CD8+ T cells in the input cell composition is adjusted or altered compared to the ratio of the naive-like CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

6. A method for generating a cell composition, the method comprising:

determining the number, number per volume, number per weight, and/or percentage of nai've-like CD4+ T cells and nai've-like CD8+ T cells in a biological sample obtained from a subject or in one or more samples derived therefrom; and

producing an input cell composition comprising CD4+ T cells and CD8+ T cells in which the ratio of the nai've-like CD4+ T cells to naive-like CD8+ T cells is between or about between 2.2: 1 to 0.8: 1, inclusive, wherein said ratio in the input cell composition is adjusted or altered compared to the ratio of the nai've-like CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

7. The method of any of claims 1-6, further comprising contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

8. A method of generating a cell composition, the method comprising:

contacting an input cell composition comprising nai've-like CD4+ T cells and nai've-like

CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition,

wherein the ratio of nai've-like CD4+ T cells to nai've-like CD8+ T cells present in the input cell composition is between or about between 0.8: 1 and 2.2: 1, inclusive.

9. The method of claim 7 or claim 8, further comprising stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

10. A method of generating a cell composition, the method comprising:

combining a first cell composition comprising nai've-like CD4+ T cells with a second cell composition comprising nai've-like CD8+ T cells to produce an input cell composition in which the ratio of nai've-like CD4+ T cells to nai've-like CD8+ T cells is between or about between 0.8: 1 and 2.2: 1, inclusive;

contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and

stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

11. The method of any of claims 1-10, wherein the naive-like CD4+ and/or the naive-like CD8+ cells:

are surface positive for a marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and/or

are surface negative for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, and KLRG1; and/or

have low expression of CD95; and/or

are negative for intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10.

12. The method of any of claims 1-11, wherein the naive-like CD4+ and/or the naive-like CD8+ cells:

are surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and/or

are surface negative for a marker selected from the group consisting of CD45RO, CD56, CD62L, and KLRG1; and/or

have low expression of CD95.

13. The method of any of claims 1-12, wherein the naive-like CD4+ and/or the naive-like CD8+ cells are surface positive for CD45RA and CCR7.

14. The method of any of claims 1-12, wherein the naive-like CD4+ and/or the naive-like CD8+ cells are surface positive for CD27 and CCR7.

15. The method of any of claims 1-14, wherein the nai've-like CD4+ and the nai've-like CD8+ cells are surface positive for CD45RA, CD27 and CCR7 and are surface negative for CD45RO.

16. The method of any of claims 1-12, wherein the nai've-like CD4+ and/or the nai've-like CD8+ cells are surface positive for CCR7 and surface negative for CD62L.

17. The method of any of claims 3-16, wherein the number, number per volume, number per weight, and/or percentage of nai've-like CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of nai've-like CD8+ T cells is determined by flow cytometry.

18. The method of any of claims 8-17, wherein the ratio of nai've-like CD4+ T cells to nai've-like CD8+ T cells has been adjusted compared to the ratio of the nai've-like CD4+ T cells to nai've-like CD8+ T cells in a biological sample from the subject.

19. The method of any of claims 1-18, wherein the input cell composition comprises a ratio of nai've-like CD4+ cells to nai've-like CD8+ cells of between or about between 0.8: 1 and 2.0: 1, 0.8: 1 and 1.6: 1, 0.8: 1 and 1.4: 1, 0.8: 1 and 1.2: 1, 1.5: 1 and 2: 1, 1.6: 1 and 1.8: 1, or 1.0: 1 and 1.2: 1, each inclusive.

20. The method of any of claims 1-19, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells of or about 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.69: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3 : 1, 1.2: 1, 1.1 : 1, or 1.0: 1.

21. The method of any of claims 1-20, wherein the input cell composition comprises a ratio of nai've-like CD4+ cells to nai've-like CD8+ cells of or about 1.1 : 1.

22. The method of any of claims 1-20, wherein the input cell composition comprises a ratio of nai've-like CD4+ cells to nai've-like CD8+ cells that are surface positive for CD45RA and CCR7 of or about 1.1 : 1.

23. The method of any of claims 1-20, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD45RA and CD27 of or about 1.69: 1.

24. The method of any of claims 1-20, wherein the input cell composition comprises a ratio of naive-like CD4+ cells to naive-like CD8+ cells that are surface positive for CD27 and CCR7 of or about 1.69: 1.

25. A method for generating a cell composition, the method comprising:

combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition in which the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells is between or about between 0.8: 1 and 2.2: 1, inclusive.

26. The method of claim 25, wherein the first cell composition is produced by isolating CD4+ T cells from a biological sample obtained from a subject and/or the second cell composition is produced by isolating CD8+ T cells from the biological sample obtained from the subject.

27. The method of claim 25 or claim 26, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the CCR7+CD45RA+CD4+ T cells in the first cell composition and/or the number, number per volume, number per weight, and/or percentage of the CCR7+CD45RA+CD8+ T cells in the second cell composition.

28. The method of any of claims 25-27, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the CCR7+CD45RA+CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of CCR7+CD45RA+CD8+ T cells in the biological sample from the subject.

29. The method of any of claims 25-28 wherein the ratio of the

CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in the input cell composition is adjusted or altered compared to the ratio of the CCR7+CD45RA+CD4+ T cells to

CCR7+CD45RA+CD8+ T cells in the biological sample from the subject.

30. A method for generating a cell composition, the method comprising:

determining the number, number per volume, number per weight, and/or percentage of

CCR7+CD45RA+CD4+ T cells and CCR7+CD45RA+CD8+ T cells in a biological sample obtained from a subject or in one or more samples derived therefrom; and

producing an input cell composition comprising CD4+ T cells and CD8+ T cells in which the ratio of the CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells is between or about between 2.2: 1 to 0.8: 1, inclusive, wherein said ratio in the input cell composition is adjusted or altered compared to the ratio of the CCR7+CD45RA+CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

31. The method of any of claims 30, further comprising contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

32. A method of generating a cell composition, the method comprising:

contacting an input cell composition comprising CCR7+CD45RA+CD4+ T cells and

CCR7+CD45RA+CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells present in the input cell composition is between or about between 0.8: 1 and 2.2: 1, inclusive.

33. The method of claim 32, further comprising stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

34. A method of generating a cell composition, the method comprising: combining a first cell composition comprising CCR7+CD45RA+CD4+ T cells with a second cell composition comprising CCR7+CD45RA+CD8+ T cells to produce an input cell composition in which the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells is between or about between 0.8: 1 and 2.2: 1, inclusive;

contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and

stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

35. The method of any of claims 25-34, wherein the number, number per volume, number per weight, and/or percentage of CCR7+CD45RA+CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of CCR7+CD45RA+CD8+ T cells is determined by flow cytometry.

36. The method of any of claims 32-35, wherein the ratio of CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells has been adjusted compared to the ratio of the CCR7+CD45RA+CD4+ T cells to CCR7+CD45RA+CD8+ T cells in a biological sample from the subject.

37. The method of any of claims 25-36, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of between or about between 0.8: 1 and 2.0: 1, 0.8: 1 and 1.6: 1, 0.8: 1 and 1.4: 1, 0.8: 1 and 1.2: 1, or 1.0: 1 and 1.2: 1, each inclusive.

38. The method of any of claims 25-37, wherein the input cell composition comprises a ratio of CCR7+CD45RA+ CD4+ cells to CCR7+CD45RA+ CD8+ cells of or about 1.5: 1, 1.4: 1, 1.3 : 1, 1.2: 1, 1.1 : 1, or 1.0: 1.

39. The method of any of claims 25-38, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of or about 1.1 : 1.

40. A method for generating a cell composition, the method comprising:

combining a first cell composition comprising CD27+CCR7+CD4+ T cells with a second cell composition comprising CD27+CCR7+CD8+ T cells to produce an input cell composition in which the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells is between or about between 1.2: 1 and 2.4: 1, inclusive.

41. The method of claim 40, wherein the first cell composition is produced by isolating CD4+ T cells from a biological sample obtained from a subject and/or the second cell composition is produced by isolating CD8+ T cells from the biological sample obtained from the subject.

42. The method of claim 40 or claim 41, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the CD27+CCR7+CD4+ T cells in the first cell composition and/or the number, number per volume, number per weight, and/or percentage of the CD27+CCR7+CD8+ T cells in the second cell composition.

43. The method of any of claims 40-42, wherein, prior to the combining, the method comprises determining the number, number per volume, number per weight, and/or percentage of the CD27+CCR7+CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of CD27+CCR7+CD8+ T cells in the biological sample from the subject.

44. The method of any of claims 40-43, wherein the ratio of the CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in the input cell composition is adjusted or altered compared to the ratio of the CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in the biological sample from the subject.

45. A method for generating a cell composition, the method comprising:

determining the number, number per volume, number per weight, and/or percentage of CD27+CCR7+CD4+ T cells and CD27+CCR7+CD8+ T cells in a biological sample obtained from a subject or in one or more samples derived therefrom; and

producing an input cell composition comprising CD4+ T cells and CD8+ T cells in which the ratio of the CD27+CCR7+CD4+ T cells to naive-like CD8+ T cells is between or about between 2.2: 1 to 0.8: 1, inclusive, wherein said ratio in the input cell composition is adjusted or altered compared to the ratio of the CD27+CCR7+CD4+ T cells to naive-like CD8+ T cells in the biological sample from the subject.

46. The method of any of claims 40-45, further comprising contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

47. A method of generating a cell composition, the method comprising:

contacting an input cell composition comprising CD27+CCR7+CD4+ T cells and

CD27+CCR7+CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition,

wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells present in the input cell composition is between or about between 0.8: 1 and 2.2: 1, inclusive.

48. The method of claim 47, further comprising stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

49. A method of generating a cell composition, the method comprising:

combining a first cell composition comprising CD27+CCR7+ CD4+ T cells with a second cell composition comprising CD27+CCR7+CD8+ T cells to produce an input cell composition in which the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+ CD8+ T cells is between or about between 0.8: 1 and 2.2: 1, inclusive;

contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and

stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

50. The method of any of claims 40-49, wherein the number, number per volume, number per weight, and/or percentage of CD27+CCR7+CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of CD27+CCR7+CD8+ T cells is determined by flow cytometry.

51. The method of any of claims 40-50, wherein the ratio of CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells has been adjusted compared to the ratio of the

CD27+CCR7+CD4+ T cells to CD27+CCR7+CD8+ T cells in a biological sample from the subject.

52. The method of any of claims 40-51, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of between or about between 0.8: 1 and 2.0: 1, 0.8: 1 and 1.6: 1, 0.8: 1 and 1.4: 1, 0.8: 1 and 1.2: 1, or 1.0: 1 and 1.2: 1, each inclusive.

53. The method of any of claims 40-52, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.5: 1, 1.4: 1, 1.3 : 1, 1.2: 1, 1.1 : 1, or 1.0: 1.

54. The method of any of claims 40-53, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.1 : 1.

55. The method of any of claims 40-53, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.69: 1.

56. A method for generating a cell composition, the method comprising:

combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition in which the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells is between or about between 0.5: 1 and 2: 1, inclusive.

57. A method for generating a cell composition, the method comprising:

determining the number, number per volume, number per weight, and/or percentage of

CD62L-CCR7+CD4+ T cells and CD62L-CCR7+CD8+ T cells in a biological sample obtained from a subject or in one or more samples derived therefrom; and

producing an input cell composition comprising CD4+ T cells and CD8+ T cells in which the ratio of the CD62L-CCR7+CD4+ T cells to naive-like CD8+ T cells is between or about between 0.5: 1 and 2: 1, inclusive, wherein said ratio in the input cell composition is adjusted or altered compared to the ratio of the CD62L-CCR7+CD4+ T cells to naive-like CD 8+ T cells in the biological sample from the subject.

58. The method of claims 56 or 57, further comprising contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the input cell composition.

59. A method of generating a cell composition, the method comprising:

combining a first cell composition comprising CD62L-CCR7+CD4+ T cells with a second cell composition comprising CD62L-CCR7+CD8+ T cells to produce an input cell composition in which the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells is between or about between 0.5: 1 and 2: 1, inclusive;

contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and

stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

60. The method of claim 58 or 59, wherein the number, number per volume, number per weight, and/or percentage of CD62L-CCR7+CD4+ T cells and/or the number, number per volume, number per weight, and/or percentage of CD62L-CCR7+CD8+ T cells is determined by flow cytometry.

61. The method of any of claims 57-60, wherein the ratio of CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells has been adjusted compared to the ratio of the CD62L-CCR7+CD4+ T cells to CD62L-CCR7+CD8+ T cells in a biological sample from the subject.

62. The method of any of claims 57-61, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of between or about between 0.5: 1 and 1.5: 1, 1 : 1 and 2: 1, 0.75: 1 and 1.5: 1, or 0.8: 1 and 1.2: 1, each inclusive.

63. The method of 57-62, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of or about 1.2: 1, 1.1 : 1, 1.0: 1, 0.9: 1, or 0.8: 1.

64. The method of any of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58, or 61-63, wherein the biological sample is or is obtained from a blood, plasma or serum sample.

65. The method of any of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58, or 61-64, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

66. The method of any of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58, or 61-65, wherein the biological sample is or is obtained from an apheresis or leukapheresis sample.

67. The method of any of claims 2-9, 18-24, 26-33, 35-39, 41-48, 51-54, 57-58, or 61-66, wherein the subject is a human subject.

68. The method of any of claims 1-67, wherein the input cell composition comprises from or from about 1 x 107 to 5 x 109 total cells or total T cells, from or from about 5 x 107 to 1 x 109 total cells or total T cells, from or from about 1 x 108 to 5 x 108 total cells or total T cells, or from or from about 2 x 108 to 5 x 108 total cells or total T cells, or of viable populations of any of the foregoing.

69. The method of any of claims 1-68, wherein the input cell composition comprises at least or at least about 1 x 108, 2 x 108, 3 x 108, 4 x 108, or 5 x 108 total cells or total T cells or a viable population of any of the foregoing.

70. The method of any of claims 9-24, 33-39, 48-54, 59-69, wherein the one or more stimulating agent is capable of activating T cells, CD4+ T cells and/or CD8+ T cells; is capable of inducing a signal through a TCR complex; and/or is capable of inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells.

71. The method of any of claims 9-24, 33-39, 48-54, 59-70, wherein the one or more stimulating agent comprises a primary agent that binds to a member of a TCR complex, optionally that specifically binds to CD3.

72. The method of claim 71, wherein the one or more stimulating agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule.

73. The method of claim 72, wherein the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

74. The method of claim 72 or claim 73, wherein the primary and secondary agents comprise antibodies, optionally wherein the one or more stimulating agent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody.

75. The method of any of claims 9-24, 33-39, 48-54, 59-74, wherein the one or more stimulating agents are present on the surface of a solid support, optionally a bead.

76. The method of claim 75, wherein the one or more stimulating agent are present on the surface of a bead and the bead is a paramagnetic bead.

77. The method of any of claims 9-24, 33-39, 48-54, 59-76, wherein the one or more one stimulating agent is selected from the group consisting of CD3-binding molecules; CD28-binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7, a vaccine comprising an antigen specifically recognized by the antigen receptor, and an anti-idiotype antibody that specifically binds the antigen receptor or combinations thereof.

78. The method of any of claims 9-24, 33-39, 48-54, 59-77, wherein the incubation is carried out for 2 to 15 days, 2 to 12 days, 2 to 12 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 4 to 12 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 6 to 12 days, 6 to 10 days, 6 to 8 days, 8 to 12 days, 8 to 10 days, or 10 to 12 days.

79. The method of any of claims 9-24, 33-39, 48-54, 59-78, wherein the incubation is carried out for at least or about at least or 4 days, 6 days, 8 days, 10 days or 12 days.

80. The method of any of claims 7-24, 31-40, 46-55, or 59-79, wherein the agent comprising the nucleic acid molecule is a viral vector or is a transposon.

81. The method of claim 80, wherein the agent comprising the nucleic acid molecule is a viral vector and the viral vector is a retroviral vector.

82. The method of claim 81, wherein the viral vector is a lentiviral vector or a gammaretroviral vector.

83. The method of any of claims 7-24, 31-39, 46-54, or 58-82, wherein the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and/or expressed on a cell or tissue of a disease, disorder or condition.

84. The method of claim 83, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer.

85. The method of claim 83 or claim 84, wherein the target antigen is a tumor antigen.

86. The method of any of claims 83-85, wherein the target antigen is selected from among Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CA9, also known as CAIX or G250), Her2/neu (receptor tyrosine kinase erbB2), CD 19, CD20, CD22, mesothelin (MSLN), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, chondroitin sulfate proteoglycan 4 (CSPG4), EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin receptor A2 (EPHa2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, type III epidermal growth factor receptor mutation (EGFR vIII), folate binding protein (FBP), Fc receptor like 5 (FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor(fetal AchR), ganglioside GD2, ganglioside GD3, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), kinase insert domain receptor (kdr), kappa light chain, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Ll-cell adhesion molecule (Ll-CAM), Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE- A 10, Preferentially expressed antigen of melanoma (PRAME), survivin, tumor-associated glycoprotein 72

(TAG72), B7-H3, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), Human high molecular weight-melanoma-associated antigen (HMW-MAA), CD171, folate receptor-alpha, CD44v7/8, ανβό integrin (avb6 integrin), 8H9, neural cell adhesion molecule (NCAM), vascular endothelial growth factor receptor (VEGF receptors or VEGFR), Trophoblast glycoprotein (TPBG also known as 5T4), KG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), natural killer group 2 member D ( KG2D) ligands, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), melan A (MART-1), glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), oncofetal antigen, TAG72, Tyrosinase related protein 1 (TRPl, also known as

TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor 2 (VEGF-R2), carcinoembryonic antigen (CEA), estrogen receptor, progesterone receptor, a prostate specific antigen, ephrinB2, CD 123, CD 133, c-Met, O-acetylated GD2 (OGD2), CE7 epitope of Ll-CAM, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD 138, a pathogen-specific antigen or pathogen-expressed antigen, and an antigen associated with a universal tag.

87. The method of any of claims 7-24, 31-39, 46-55, or 58-86, wherein the recombinant receptor is or comprises a functional non-TCR antigen receptor or a TCR or antigen-binding fragment thereof88. The method of any of claims 7-24, 31-39, 46-55, or 58-86, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

89. The method of claim 88, wherein the chimeric antigen receptor comprises an extracellular domain comprising an antigen-binding domain that specifically binds to an antigen and an intracellular signaling domain comprising an IT AM.

90. The method of claim 89, wherein the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment.

91. The method of claim 89 or claim 90, wherein the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (Οϋ3ζ) chain, or a signaling portion thereof.

92. The method of any of claims 89, 90 and 91, wherein the intracellular signaling region further comprises a costimulatory signaling region.

93. The method of claim 92, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

94. The method of claim 92 or claim 93, wherein the costimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4- IBB or an ICOS or a signaling portion thereof.

95. The method of any of claims 88-93, wherein:

the CAR comprises an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain and optionally further comprises a spacer between the transmembrane domain and the scFv;

the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain; or

the CAR comprises, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4- IBB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain.

96. The method of any of claims 2-9, 11-24, 26-33, 35-39, 41-48, 51-55, 57-58, or 61-95, wherein the subject has a disease or condition, optionally wherein the recombinant receptor specifically recognizes or specifically bind to an antigen associated with, or expressed or present on cells of, the disease or condition.

97. The method of any of claims 1-96, wherein the method produces an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, varies by no more than 20% or no more than 10% or no more than 5% from an average of said ratio in a plurality of T cell compositions produced by the method and/or varies from such average by no more than one standard deviation.

98. The method of any of claims 1-97, wherein the method produces an output composition in which the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, is between at or about 0.5: 1 and 2: 1 or 0.8: 1 and 1.6: 1 or 1 : 1 and 1.5: 1, each inclusive.

99. The method of any of claim 97 or claim 98, wherein the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, in the output composition is or is about 1.2: 1, 1.1 : 1, 1 : 1, 0.9: 1, or 0.8: 1.

100. The method of any of claims 97-99, wherein the ratio of recombinant receptor-expressing CD4+ T cells to recombinant receptor-expressing CD8+ T cells, optionally the ratio of viable cells thereof, in the output composition is or is about 1 : 1.

101. An output composition produced by the method of any of claims 1-100.

102. A pharmaceutical composition comprising the output composition of claim 101.

103. The pharmaceutical composition of claim 102, further comprising a

pharmaceutical carrier.

104. A method of treatment, comprising administering to a mammalian subject an output composition produced by the method of any of claims 1-100 or a pharmaceutical composition of claim 102 or claim 103.

105. The method of claim 104, wherein the cells are derived from the subject to which the cells are administered.