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1. WO2020112860 - METHODS OF IDENTIFYING IMMUNOMODULATORY GENES

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[ EN ]

CLAIMS

What is claimed is:

1. A method of screening a plurality of single candidate genes, said method comprising:

a. expressing an exogenous cellular receptor, or a functional fragment

thereof, in a plurality of separate populations of immune cells, wherein each population comprises a plurality of immune cells;

b. introducing into each of said separate populations of immune cells a

CRISPR system that comprises:

i. a guide nucleic acid that binds a portion of a single candidate gene, wherein said single candidate gene is different for each of said separate populations of immune cells; and

ii. an exogenous nuclease, or a nucleic acid encoding said exogenous nuclease;

thereby generating a plurality of separate populations of engineered immune cells that comprise a genomic disruption in said single candidate gene, wherein said genomic disruption that suppresses expression of said single candidate gene;

c. performing an in vitro assay that comprises contacting said plurality of engineered immune cells with a plurality of cells expressing a cognate antigen of said exogenous cellular receptor or said functional fragment thereof in vitro; and

d. obtaining a readout from said in vitro assay, to thereby determine an effect of said genomic disruption that suppresses expression of said single candidate gene on said plurality of separate populations of engineered immune cells.

2. The method of claim 1, wherein said readout comprises determining a level of cytolytic activity of each of said plurality of separate populations of engineered immune cells.

3. The method of claim 2, wherein said level of cytolytic activity is determined by a chromium release assay, an electrical impedance assay, time-lapse microscopy, or a co-culture assay.

4. The method of claim 1, wherein said readout comprises determining a level of proliferation of each of said plurality of separate populations of engineered immune cells.

5. The method of claim 4, wherein said level of proliferation is determined by a Carboxyfluorescein Succinimidyl Ester (CFSE) assay, microscopy, an electrical impedance assay, or flow cytometry.

6. The method of claim 1, wherein said readout comprises determining a level of a factor expressed by each of said plurality of separate populations of engineered immune cells.

7. The method of claim 6, wherein said factor is a protein.

8. The method of claim 7, wherein said protein is secreted from said population of engineered immune cells.

9. The method of claim 7 or 8, wherein said protein is a cytokine or chemokine.

10. The method of claim 7, wherein said protein is a cell surface protein.

11. The method of any one of claims 6-10, wherein said expression is determined by flow cytometry, western blot, or ELISA.

12. The method of preceding claim, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of immune cells of each of said separate populations of immune cells comprise said genomic disruption, in the absence of a selection step.

13. The method of claim 12, wherein at least 80% of immune cells of each of said separate populations of immune cells comprise said genomic disruption, in the absence of a selection step.

14. The method of claim 12, wherein at least 90% of immune cells of each of said separate populations of immune cells comprise said genomic disruption, in the absence of a selection step.

15. The method of any one of claims 12-14, wherein said percentage of immune cells of each of said separate populations of immune cells is determined by Tracking of Indels by

Decomposition (TIDE) analysis.

16. The method of any preceding claim, wherein said exogenous cellular receptor is integrated into the genome of said plurality of separate populations of immune cells.

17. The method of claim 16, wherein said exogenous cellular receptor is integrated into an endogenous gene sequence that encodes an endogenous cellular receptor.

18. The method of claim 16, wherein said exogenous cellular receptor is integrated into a safe harbor site.

19. The method of claim 18, wherein said safe harbor site is an AAVS site (e.g., AAVS1, AAVS2), CCR5, or hROSA26.

20. The method of claim 16, wherein said exogenous cellular receptor is integrated into a portion of a gene that encodes a protein that functions as a negative regulator of an immune response of said plurality of immune cells.

21. The method of any preceding claim, wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of immune cells of each of said separate populations of immune cells express said exogenous cellular receptor, in the absence of a selection step.

22. The method of claim 21, wherein at least 70% of immune cells of each of said separate populations of immune cells express said exogenous cellular receptor, in the absence of a selection step.

23. The method of claim 21, wherein at least 80% of immune cells of each of said separate populations of immune cells express said exogenous cellular receptor, in the absence of a selection step.

24. The method of claim 21, wherein at least 90% of immune cells of each of said separate populations of immune cells express said exogenous cellular receptor, in the absence of a selection step.

25. The method of any one of claims 21-24, wherein said percentage of immune cells of each of said separate populations of immune cells is determined by flow cytometry or sequencing.

26. The method of any preceding claim, wherein said genomic disruption is a double strand break.

27. The method of any preceding claim, wherein said nuclease is introduced using

electroporation.

28. The method of any preceding claim, wherein said nuclease is an endonuclease.

29. The method of claim 28, wherein said endonuclease is selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3,

Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, and Cas9HiFi.

30. The method of claim 29, wherein said endonuclease is Cas9.

31. The method of preceding claim, wherein said guide nucleic acid is a guide ribonucleic acid (gRNA).

32. The method of any preceding claim, wherein said guide nucleic acid comprises a phosphorothioate (PS) linkage, a 2’-fluoro (2’-F) modification, a 2’-0-methyl (2’-0-Me) linkage, a 2-O-Methyl 3phosphorothioate linkage, a S-constrained ethyl (cEt) modification, or any combination thereof

33. The method of any preceding claim, wherein said guide nucleic acid is introduced using electroporation.

34. The method of any preceding claim, wherein said exogenous cellular receptor is introduced using electroporation.

35. The method of any one of claims 1-33, wherein said exogenous cellular receptor is introduced using a viral vector.

36. The method of claim 35, wherein said viral vector is an adeno-associated virus (AAV) vector.

37. The method of claim 36, wherein said AAV vector is selected from the group consisting of a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self complementary AAV (scAAV) vector, a modified AAV vector, and any combination thereof.

38. The method of claim 37, wherein said AAV vector is a chimeric AAV vector.

39. The method of claim 38, wherein said chimeric AAV vector comprises a modification in at least one AAV capsid gene sequence.

40. The method of any preceding claim, wherein said exogenous cellular receptor is a T-cell receptor (TCR), B cell receptor (BCR), NK cell receptor, dendritic cell receptor, monocyte receptor, macrophage receptor, neutrophil receptor, eosinophil receptor, or a chimeric antigen receptor (CAR).

41. The method of claim 40, wherein said exogenous cellular receptor is a T-cell receptor (TCR).

42. The method of any preceding claim, wherein said single gene is an immunomodulatory gene.

43. The method of any preceding claim, wherein said single gene is a candidate immune checkpoint gene.

44. The method of any preceding claim, further comprising cryopreserving said separate populations of engineered immune cells.

45. The method of any preceding claim, further comprising processing said readout to identify a candidate immunomodulatory gene.

46. The method of claim 45, wherein said processing comprises determining a criterion from at least one of: cytolytic activity, gene expression of said candidate immunomodulatory gene, intracellular location of a protein encoded by said candidate immunomodulatory gene, loss-of-function association with a human disease of said candidate immunomodulatory gene, a guide nucleic acid score of a guide nucleic acid that binds to a portion of said candidate

immunomodulatory gene, existing drugs in development that target said candidate

immunomodulatory gene, existing drugs that target said candidate immunomodulatory gene, or loss-of-function phenotype of said candidate immunomodulatory gene, or any combination thereof.

47. The method of claim 46, wherein said processing comprises determining a criterion from at least two, three, four, five, six, seven, or eight of: cytolytic activity, gene expression of said candidate immunomodulatory gene, intracellular location of a protein encoded by said candidate immunomodulatory gene, loss-of-function association with a human disease of said candidate immunomodulatory gene, a guide nucleic acid score of a guide nucleic acid that binds to a portion of said candidate immunomodulatory gene, existing drugs in development that target said candidate immunomodulatory gene, existing drugs that target said candidate immunomodulatory gene, or loss-of-function phenotype of said candidate immunomodulatory gene, or any combination thereof.

48. The method of claim 46, wherein said processing comprises ranking at least two candidate immunomodulatory genes according to said at least one criterion to produce ranked candidate immunomodulatory genes.

49. The method of claim 47, wherein said processing comprises ranking at least two candidate immunomodulatory genes according to said at least two, three, four, five, six, seven, or eight criterion to produce ranked candidate immunomodulatory genes.

50. The method of claim 46 or 48, wherein said processing comprises ranking at least 10,

100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 candidate immunomodulatory genes according to said at least one criterion.

51. The method of claim 47 or 49, wherein said processing comprises ranking at least 10,

100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 candidate immunomodulatory genes according to said at least two, three, four, five, six, seven, or eight criterion.

52. The method of claim 48 or 49, further comprising selecting a top 10, 20, 30, 40, or 50 of said ranked candidate immunomodulatory genes to thereby generate a ranked output.

53. The method of claim 52, further comprising identifying at least one of a gene family, a gene function, or an intracellular signaling pathway from said ranked output, to thereby generate an analyzed ranked output.

54. The method of claim 53, further comprising correlating cytolytic activity of said analyzed ranked output, to thereby generate a cytolytic-correlated ranked output.

55. The method of claim 54, further comprising ranking said candidate immunomodulatory genes from said cytolytic-correlated ranked output according to said intracellular location of a protein encoded by said candidate immunomodulatory gene.

56. The method of claim 54, further comprising ranking said candidate immunomodulatory genes from said cytolytic-correlated ranked output according to said existing drug in

development that targets said candidate immunomodulatory gene and said existing drug against said candidate immunomodulatory gene.

57. The method of any preceding claim, wherein each of said populations of engineered immune cells comprises a plurality of T cells, tumor infiltrating lymphocytes (TILs), NK cells, B cell, dendritic cells, monocytes, macrophages, neutrophils, or eosinophils.

58. The method of claim 57, wherein each of said populations of engineered immune cells comprises a plurality of T cells.

59. The method of claim 58, wherein said plurality of T cells comprises a plurality of CD8+ T cells.

60. The method of claim 58, wherein said plurality of T cells comprises a plurality of CD4+ T cells.

61. The method of claim 58, wherein said plurality of T cells comprises a plurality of CD4+ T cells and CD8+ T cells.

62. The method of any preceding claim, wherein each of said populations of engineered immune cells comprises a plurality of human cells.

63. The method of any preceding claim, wherein each of said populations of engineered immune cells comprises a plurality of primary cells.

64. The method of any preceding claim, wherein each of said populations of engineered immune cells comprises a plurality of ex vivo cells.

65. The method of any preceding claim, wherein said plurality of separate populations of immune cells comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate populations of immune cells.

66. The method of any preceding claim, wherein said each of said populations of engineered immune cells comprises a transgene that encodes for a protein that improves immunomodulatory function of said engineered immune cells.

67. The method of claim 66, wherein said transgene is integrated in the genome of said engineered immune cells.

68. The method of claim 67, wherein said transgene is integrated into a safe harbor site.

69. The method of claim 68, wherein said safe harbor site is site is an AAVS site (e.g., AAVS1, AAVS2), CCR5, or hROSA26.

70. The method of claim 67, wherein said transgene is integrated into a portion of a gene that encodes a protein that functions as a negative regulator of an immune response of said plurality of immune cells.

71. The method of any preceding claim, wherein said each of said populations of engineered immune cells comprises a genetic modification that enhances expression of a gene that encodes for a protein that improves immunomodulatory function of said engineered immune cells

72. The method of claim 71, wherein said plurality of cells that express said cognate antigen are cancer cells.

73. The method of claim 71 or 72, wherein said cancer cells are primary cancer cells or from a cancer cell line.

74. The method of any one of claims 71-73, wherein said cancer cells comprise a genomic disruption in at least one gene.

75. The method of claim 74, wherein said genomic disruption is mediated by a CRISPR system that comprises a gRNA that binds to a portion of said gene and a nuclease that mediates cleavage of genomic DNA.

76. The method of claim 74 or 75, wherein said genomic disruption is a double strand break.

77. The method of any one of claims 74-76, wherein said at least one gene encodes a protein that that a negative regulator of an immune response.

78. The method of claim 77, wherein said protein is a ligand of a checkpoint inhibitor.

79. The method of claim 78, wherein said protein is a ligand of a checkpoint inhibitor selected from the group consisting of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 10 receptor subunit alpha (ILIORA), interleukin 10 receptor subunit beta (IL10RB), adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), indoleamine 2,3-dioxygenase 1 (IDOl), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site l(AAVSl), or chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5), CD160 molecule (CD160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor l(LAIRl), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a

(TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto oncogene (SKIL), TGFB induced factor homeobox l(TGIFl), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1(SIT1), forkhead box P3(FOXP3), PR domain l(PRDMl), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2(GUCY1A2), guanylate cyclase 1, soluble, alpha 3(GUCY1A3), guanylate cyclase 1, soluble, beta 2(GUCY1B2), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or guanylate cyclase 1, soluble, beta 3(GUCY1B3), egl-9 family hypoxia-inducible factor 1 ( EGLN1), egl-9 family hypoxia-inducible factor 2 (EGLN2), egl-9 family hypoxia-inducible factor 3 (EGLN3), protein phosphatase 1 regulatory subunit 12C (PPP1R12C), NAD-dependent deacetylase sirtuin 2 (SIRT2), and Protein Tyrosine Phosphatase Non-Receptor Type 1 (PTPN1).

80. The method of any one of claims 72-79, wherein said cancer cells express at least one exogenous protein.

81. The method of claim 80, wherein said exogenous protein is a cell surface receptor.

82. The method of claim 80, wherein said exogenous protein is an intracellular protein.

83. The method of any one of claims 80-82, wherein a transgene encoding said exogenous protein is integrated into the genome of said cancer cells.

84. The method of any one of claims 80-83, wherein said exogenous protein modulates the ability of an immune cell to recognize and/or kill said cancer cells.

85. The method of any preceding claim, wherein each of said separate populations of immune cells are contained with separate compartments of one or more arrays.

86. A composition comprising a plurality of separate populations of immune cells, wherein each separate population of immune cells comprises a plurality of immune cells that i) express an exogenous cellular receptor; and ii) comprise a CRISPR system that comprises a guide nucleic acid that binds a portion of a single candidate gene, wherein said single candidate gene is different for each of said separate populations of immune cells; and an exogenous nuclease, or a nucleic acid encoding said exogenous nuclease.

87. The composition of claim 86, wherein said population of said plurality of immune cells of each separate population comprises a genomic disruption in said single candidate gene.

88. The composition of claim 87, wherein at least 70%, 80%, or 90% of said plurality of immune cells of each separate population comprises a genomic disruption in said single candidate gene.

89. The composition of any one of claims 86-88, wherein each of said separate populations of immune cells are contained with separate compartments of one or more arrays.

90. The composition of any one of claim 86-88, wherein said plurality of separate populations of immune cells comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,

600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate populations of immune cells.

91. A composition comprising a plurality of separate cell populations that each comprise i) a plurality of immune cells that express an exogenous cellular receptor and ii) cells that express a cognate antigen of said exogenous cellular receptor; wherein each of said plurality of immune cells comprises an altered genome sequence of a single candidate gene, and wherein said single candidate gene is different for each of said separate cell populations.

92. The composition of claim 91, wherein at least 70%, 80%, or 90% of said plurality of immune cells of each separate cell population comprises said altered genome sequence of said single candidate gene.

93. The composition of claim 91 or 92, wherein each of said separate cell populations are contained with separate compartments of one or more arrays.

94. The composition of any one of claims 91-93, wherein said plurality of separate cell populations comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,

700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate cell populations.

95. A method of screening a plurality of single candidate genes, said method comprising:

a. obtaining a plurality of separate populations of cancer cells that express an antigen, wherein each population comprises a plurality of cancer cells; b. introducing into each of said separate populations of cancer cells a

CRISPR system that comprises:

i. a guide nucleic acid that binds a portion of a single candidate gene, wherein said single candidate gene is different for each of said separate populations of cancer cells; and

ii. an exogenous nuclease, or a nucleic acid encoding said exogenous nuclease;

thereby generating a plurality of separate populations of engineered cancer cells that comprise a genomic disruption in said single candidate gene, wherein said genomic disruption suppresses expression of said single candidate gene;

c. performing an in vitro assay that comprises contacting in vitro said

plurality of engineered cancer cells with a plurality of immune cells that express a cellular receptor, or functional fragment thereof, that binds to said antigen; and

d. obtaining a readout from said in vitro assay, to thereby determine an effect of said genomic disruption that suppresses expression of said single candidate gene on said plurality of separate populations of engineered cancer cells or said immune cells that express a cellular receptor, or functional fragment thereof, that binds to said antigen.

96. The method of claim 95, wherein said readout comprises determining a level of cell death of each of said separate populations of engineered cancer cells.

97. The method of claim 96, wherein said level of cell death is determined by flow cytometry or microscopy.

98. The method of claim 95, wherein said readout comprises determining a time to which a certain percentage of cells each of said separate populations of engineered cancer cells are killed.

99. The method of claim 98, wherein said level of cell death is determined by flow cytometry or microscopy.

100. The method of claim 95, wherein said readout comprises determining a level of cytolytic activity of said plurality of immune cells.

101. The method of claim 100, wherein said level of cytolytic activity is determined by a chromium release assay, an electrical impedance assay, time-lapse microscopy, or a co-culture assay.

102. The method of claim 95, wherein said readout comprises determining a level of proliferation of said plurality of immune cells.

103. The method of claim 102, wherein said level of proliferation is determined by a

Carboxyfluorescein Succinimidyl Ester (CFSE) assay, microscopy, an electrical impedance assay, or flow cytometry.

104. The method of claim 95, wherein said readout comprises determining a level of a factor expressed by said plurality of immune cells.

105. The method of claim 104, wherein said factor is a protein.

106. The method of claim 105, wherein said protein is secreted from said population of engineered immune cells.

107. The method of claim 104 or 105, wherein said protein is a cytokine or chemokine.

108. The method of claim 104, wherein said protein is a cell surface protein.

109. The method of any one of claims 104-108, wherein said expression is determined by flow cytometry, western blot, or ELISA.

110. The method of any one of claims 95-109, wherein said antigen is an endogenous antigen.

111. The method of any one of claims 95-109, wherein said antigen is an exogenous antigen.

112. The method of claim 111, wherein step a. comprises expressing said exogenous antigen in each of said separate populations of cancer cells.

113. The method of any one of claims 95-112, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of cancer cells of each of said separate populations of cancer cells comprise said genomic disruption, in the absence of a selection step.

114. The method of claim 113, wherein at least 80% of cancer cells of each of said separate populations of cancer cells comprise said genomic disruption, in the absence of a selection step.

115. The method of claim 113, wherein at least 90% of cancer cells of each of said separate populations of cancer cells comprise said genomic disruption, in the absence of a selection step.

116. The method of any one of claims 113-115, wherein said percentage of cancer cells of each of said separate populations of cancer cells is determined by Tracking of Indels by

Decomposition (TIDE) analysis.

117. The method of any one of claims 95-116, wherein said cellular receptor is an

immunomodulatory cellular receptor.

118. The method of any one of claims 95-117, wherein said cellular receptor is an exogenous cellular receptor.

119. The method of claim 118, wherein said exogenous cellular receptor is integrated into the genome of said plurality of immune cells.

120. The method of claim 119, wherein said exogenous cellular receptor is integrated into an endogenous gene sequence that encodes an exogenous cellular receptor.

121. The method of claim 119, wherein said exogenous cellular receptor is integrated into a safe harbor site.

122. The method of claim 121, wherein said safe harbor site is site is an AAVS site (e.g., AAVS1, AAVS2), CCR5, or hROSA26.

123. The method of claim 120, wherein said exogenous cellular receptor is integrated into a portion of a gene that encodes a protein that functions as a negative regulator of an immune response of said plurality of immune cells.

124. The method of claim 123, wherein said integration decreases or inhibits expression of said protein that functions as a negative regulator of an immune response of said plurality of immune cells.

125. The method of claim 123 or 124, wherein said gene encodes for a protein selected from the group consisting of CISH, PD1, CTLA4, adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), indoleamine 2,3 -di oxygenase 1 (IDOl), killer cell immunoglobulin-like receptor, three domains, long cytoplas ic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site l(AAVSl), or chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5), CD160 molecule (CD160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor l(LAIRl), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto oncogene (SKIL), TGFB induced factor homeobox l(TGIFl), programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 10 receptor subunit alpha (ILIORA), interleukin 10 receptor subunit beta (ILIORB), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1(SIT1), forkhead box P3 (FOXP3), PR domain l(PRDMl), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), guanylate cyclase 1, soluble, alpha 3 (GUCY1A3), guanylate cyclase 1, soluble, beta 2(GUCY1B2), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or guanylate cyclase 1, soluble, beta 3 (GUCY1B3), egl-9 family

hypoxia-inducible factor 1 (EGLN1), egl-9 family hypoxia-inducible factor 2 (EGLN2), egl-9 family hypoxia-inducible factor 3 (EGLN3), protein phosphatase 1 regulatory subunit 12C (PPP1R12C), NAD-dependent deacetylase sirtuin 2 (SIRT2), or Protein Tyrosine Phosphatase Non-Receptor Type 1 (PTPN1).

126. The method of any one of claims 95-125, wherein at least 50%, 60%, 65%, 70%, 75%,

80%, 85%, 90%, 95%, or 99% of said plurality of immune cells express said cellular receptor, in the absence of a selection step.

127. The method of claim 126, wherein said percentage of immune cells of said plurality of immune cells is determined by flow cytometry or sequencing.

128. The method of any one of claims 95-127, wherein said genomic disruption is a double strand break.

129. The method of any one of claims 95-128, wherein said nuclease is introduced using electroporation.

130. The method of any one of claims 95-129, wherein said nuclease is an endonuclease.

131. The method of claim 130, wherein said endonuclease is selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, and Cas9HiFi.

132. The method of claim 131, wherein said endonuclease is Cas9.

133. The method of any one of claims 95-132, wherein said guide nucleic acid is a guide ribonucleic acid (gRNA).

134. The method of any one of claims 95-133, wherein said guide nucleic acid comprises a phosphorothioate (PS) linkage, a 2’-fluoro (2’-F) modification, a 2’-0-methyl (2’-0-Me) linkage, a 2-O-Methyl 3phosphorothioate linkage, a S-constrained ethyl (cEt) modification, or any combination thereof

135. The method of any one of claims 95-134, wherein said guide nucleic acid is introduced using electroporation.

136. The method of any one of claims 95-135, wherein said cellular receptor is an exogenous cellular receptor introduced using electroporation.

137. The method of any one of claims 95-135, wherein said cellular receptor is an exogenous cellular receptor introduced using a viral vector.

138. The method of claim 137, wherein said viral vector is an adeno-associated virus (AAV) vector.

139. The method of claim 138, wherein said AAV vector is selected from the group consisting of a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self complementary AAV (scAAV) vector, a modified AAV vector, and any combination thereof.

140. The method of claim 139, wherein said AAV vector is a chimeric AAV vector.

141. The method of claim 140, wherein said chimeric AAV vector comprises a modification in at least one AAV capsid gene sequence.

142. The method of any one of claims 95-141, wherein said cellular receptor is a T-cell receptor (TCR), B cell receptor (BCR), NK cell receptor, dendritic cell receptor, monocyte receptor, macrophage receptor, neutrophil receptor, eosinophil receptor, or a chimeric antigen receptor (CAR).

143. The method of claim 142, wherein said cellular receptor is a T-cell receptor (TCR).

144. The method of any one of claims 95-143, wherein said single gene is an

immunomodulatory gene.

145. The method of any one of claims 95-144, wherein said single gene is a candidate immune checkpoint receptor ligand gene.

146. The method of any one of claims 95-145, further comprising cryopreserving said separate populations of engineered cancer cells.

147. The method of any one of claims 95-146, further comprising processing said readout to identify a candidate immunomodulatory gene.

148. The method of claim 147, wherein said processing comprises determining a criterion from at least one of: cytolytic activity, gene expression of said candidate immunomodulatory gene, intracellular location of a protein encoded by said candidate immunomodulatory gene, loss-of-function association with a human disease of said candidate immunomodulatory gene, a guide nucleic acid score of a guide nucleic acid that binds to a portion of said candidate

immunomodulatory gene, existing drugs in development that target said candidate

immunomodulatory gene, existing drugs that target said candidate immunomodulatory gene, or loss-of-function phenotype of said candidate immunomodulatory gene, or any combination thereof.

149. The method of claim 148, wherein said processing comprises determining a criterion from at least two, three, four, five, six, seven, or eight of: cytolytic activity, gene expression of said candidate immunomodulatory gene, intracellular location of a protein encoded by said candidate immunomodulatory gene, loss-of-function association with a human disease of said candidate immunomodulatory gene, a guide nucleic acid score of a guide nucleic acid that binds to a portion of said candidate immunomodulatory gene, existing drugs in development that target said candidate immunomodulatory gene, existing drugs that target said candidate immunomodulatory gene, or loss-of-function phenotype of said candidate immunomodulatory gene, or any combination thereof.

150. The method of claim 148, wherein said processing comprises ranking at least two candidate immunomodulatory genes according to said at least one criterion to produce ranked candidate immunomodulatory genes.

151. The method of claim 149, wherein said processing comprises ranking at least two candidate immunomodulatory genes according to said at least two, three, four, five, six, seven, or eight criterion to produce ranked candidate immunomodulatory genes.

152. The method of claim 148 or 150, wherein said processing comprises ranking at least 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 candidate immunomodulatory genes according to said at least one criterion.

153. The method of claim 149 or 151, wherein said processing comprises ranking at least 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 candidate immunomodulatory genes according to said at least two, three, four, five, six, seven, or eight criterion.

154. The method of claim 150 or 151, further comprising selecting a top 10, 20, 30, 40, or 50 of said ranked candidate immunomodulatory genes to thereby generate a ranked output.

155. The method of claim 154, further comprising identifying at least one of a gene family, a gene function, or an intracellular signaling pathway from said ranked output, to thereby generate an analyzed ranked output.

156. The method of claim 155, further comprising correlating cytolytic activity of said analyzed ranked output, to thereby generate a cytolytic-correlated ranked output.

157. The method of claim 156, further comprising ranking said candidate immunomodulatory genes from said cytolytic-correlated ranked output according to said intracellular location of a protein encoded by said candidate immunomodulatory gene.

158. The method of claim 157, further comprising ranking said candidate immunomodulatory genes from said cytolytic-correlated ranked output according to said existing drug in

development that targets said candidate immunomodulatory gene and said existing drug against said candidate immunomodulatory gene.

159. The method of any one of claims 95-158, wherein said plurality of immune cells comprises a plurality of T cells, tumor infiltrating lymphocytes (TILs), NK cells, B cell, dendritic cells, monocytes, macrophages, neutrophils, or eosinophils.

160. The method of claim 159, wherein said plurality of immune cells comprises a plurality of T cells.

161. The method of claim 160, wherein said plurality of T cells comprises a plurality of CD8+ T cells.

162. The method of claim 160, wherein said plurality of T cells comprises a plurality of CD4+ T cells.

163. The method of claim 160, wherein said plurality of T cells comprises a plurality of CD4+ T cells and CD8+ T cells.

164. The method of any one of claims 95-163, wherein said plurality of immune cells comprises a plurality of human cells.

165. The method of any one of claims 95-164, wherein said plurality of immune cells comprises a plurality of primary cells.

166. The method of any one of claims 95-165, wherein said plurality of immune cells comprises a plurality of ex vivo cells.

167. The method of any one of claims 95-166, wherein said plurality of separate populations of cancer cells comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate populations of cancer cells.

168. The method of any one of claims 95-167, wherein said plurality of immune cells comprises a transgene that encodes for a protein that improves immunomodulatory function of said immune cells.

169. The method of claim 168, wherein said transgene is integrated in the genome of said immune cells.

170. The method of claim 169, wherein said transgene is integrated into a safe harbor site.

171. The method of claim 170, wherein said safe harbor site is site is an AAVS site (e.g., AAVS1, AAVS2), CCR5, or hROSA26.

172. The method of any one of claims 95-171, wherein said plurality of immune cells comprises a genetic modification that enhances expression of a gene that encodes for a protein that improves immunomodulatory function of said immune cells.

173. The method of claim 172, wherein said transgene is integrated into a portion of a gene that encodes a protein that functions as a negative regulator of an immune response of said immune cells.

174. The method of any one of claims 95-173, wherein each of said separate populations of cancer cells comprise a genomic disruption in at least one gene.

175. The method of claim 174, wherein said genomic disruption is mediated by a CRISPR system that comprises a gRNA that binds to a portion of said gene and a nuclease that mediates cleavage of genomic DNA.

176. The method of claim 174 or 175, wherein said genomic disruption is a double strand break.

177. The method of any one of claims 174-176, wherein said at least one gene encodes a protein that that a negative regulator of an immune response.

178. The method of claim 177, wherein said protein is a ligand of a checkpoint inhibitor.

179. The method of claim 178, wherein said protein is a ligand of a checkpoint inhibitor selected from the group consisting of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 10 receptor subunit alpha (ILIORA), interleukin 10 receptor subunit beta (IL10RB), adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), indoleamine 2,3-dioxygenase 1 (IDOl), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site l(AAVSl), or chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR5), CD160 molecule (CD160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor l(LAIRl), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto oncogene (SKIL), TGFB induced factor homeobox l(TGIFl), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1(SIT1), forkhead box P3(FOXP3), PR domain l(PRDMl), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2(GUCY1A2), guanylate cyclase 1, soluble, alpha 3(GUCY1A3),

guanylate cyclase 1, soluble, beta 2(GUCY1B2), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or guanylate cyclase 1, soluble, beta 3(GUCY1B3), egl-9 family hypoxia-inducible factor 1 ( EGLN1), egl-9 family hypoxia-inducible factor 2 (EGLN2), egl-9 family hypoxia-inducible factor 3 (EGLN3), protein phosphatase 1 regulatory subunit 12C (PPP1R12C), NAD-dependent deacetylase sirtuin 2 (SIRT2), and Protein Tyrosine Phosphatase Non-Receptor Type 1 (PTPN1).

180. The method of any one of claims 95-179, wherein said cancer cells express at least one exogenous protein.

181. The method of claim 180, wherein said exogenous protein is a cell surface receptor.

182. The method of claim 181, wherein said exogenous protein is an intracellular protein.

183. The method of any one of claims 180-182, wherein a transgene encoding said exogenous protein is integrated into the genome of said cancer cells.

184. The method of any one of claims 180-183, wherein said exogenous protein modulates the ability of an immune cell to recognize and/or kill said cancer cells.

185. The method of any one of claims 95-184, wherein each of said separate populations of immune cells are contained with separate compartments of one or more arrays.

186. A composition comprising a plurality of separate populations of cancer cells, wherein each separate population of cancer cells comprises a plurality of cancer cells that i) expresses an antigen; and ii) comprise a CRISPR system that comprises a guide nucleic acid that binds a portion of a single candidate gene, wherein said single candidate gene is different for each of said separate populations of cancer cells; and an exogenous nuclease, or a nucleic acid encoding said exogenous nuclease.

187. The composition of claim 186, wherein said population of said plurality of cancer cells of each separate population comprises a genomic disruption in said single candidate gene.

188. The composition of claim 187, wherein at least 70%, 80%, or 90% of said plurality of cancer cells of each separate population comprises a genomic disruption in said single candidate gene.

189. The composition of any one of claims 186-188, wherein each of said separate populations of cancer cells are contained with separate compartments of one or more arrays.

190. The composition of any one of claim 186-189, wherein said plurality of separate populations of cancer cells comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate populations of cancer cells.

191. A composition comprising a plurality of separate cell populations that each comprise: i) a plurality of cancer cells that express an antigen; and ii) cells that express a cellular receptor, or functional fragment thereof, that binds to said antigen; wherein each of said plurality of cancer cells comprises an altered genome sequence of a single candidate gene, and wherein said single candidate gene is different for each of said separate cell populations.

192. The composition of claim 191, wherein at least 70%, 80%, or 90% of said population of said plurality of cancer cells of each separate cell populations comprises said altered genome sequence of said single candidate gene.

193. The composition of claim 191 or 192, wherein each of said separate cell populations are contained with separate compartments of one or more arrays.

194. The composition of any one of claims 191-193, wherein said plurality of separate cell populations comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, or 100000 separate cell populations.