[go: up one dir, main page]

WO2025184170A1 - Methods and compositions for improving t cell function in an immunosuppressive environment - Google Patents

Methods and compositions for improving t cell function in an immunosuppressive environment

Info

Publication number
WO2025184170A1
WO2025184170A1 PCT/US2025/017337 US2025017337W WO2025184170A1 WO 2025184170 A1 WO2025184170 A1 WO 2025184170A1 US 2025017337 W US2025017337 W US 2025017337W WO 2025184170 A1 WO2025184170 A1 WO 2025184170A1
Authority
WO
WIPO (PCT)
Prior art keywords
immunosuppressive
lymphocyte
cells
resistance gene
modified lymphocyte
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/017337
Other languages
French (fr)
Inventor
Mateusz Legut
Neville E. SANJANA
Maria T. GUARINO
Iván REYES TORRES
Mitchell S. WANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Overt Bio Inc
Original Assignee
Overt Bio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Overt Bio Inc filed Critical Overt Bio Inc
Publication of WO2025184170A1 publication Critical patent/WO2025184170A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/36—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase

Definitions

  • the present disclosure relates in some aspects to modified lymphocytes capable of resisting immunosuppressive cellular environments.
  • Tregs regulatory T cells
  • TIL tumor-infiltrating lymphocytes
  • a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
  • the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
  • the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
  • the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46
  • a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of MCAM, FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1
  • a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3,
  • a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, COPZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, COPZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof;
  • the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
  • the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
  • a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the modified lymphocyte comprises an exogenous nucleic acid encoding LTBR.
  • the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding LTBR.
  • the increased level of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a tumour microenvironment compared to an unmodified lymphocyte.
  • the increased level of the immunosuppressive resistance gene results in increased tumor cell killing of the modified lymphocyte in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene.
  • the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6.
  • the nucleic acid further encodes at least two immunosuppressive resistance genes selected from the group consisting of FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
  • the modified immunosuppressive resistance genes selected from the group consisting of FOS
  • the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the CAR or the TCR binds to a tumor antigen.
  • the increased level of LTBR results in increased killing of cells expressing a low level of antigen in a tumor killing assay compared to a lymphocyte that does not express the LTBR, wherein the therapeutic protein is a CAR or a TCR, wherein the antigen is bound by the CAR or the TCR.
  • the tumor killing assay is an in vitro tumor killing assay.
  • the CAR or the TCR binds to HER2 or Claudin-6 (CLDN6).
  • the modified lymphocyte further comprises an expression cassette comprising the nucleic acid encoding the CAR or the TCR.
  • the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in the same expression cassette.
  • the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in separate expression cassettes.
  • exhaustion of the modified lymphocyte is reduced compared to a lymphocyte that does not express the immunosuppressive resistance gene.
  • the modified lymphocyte maintains the ability to kill tumor cells expressing a tumor antigen following at least two exposures to the tumor cells.
  • the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
  • the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs).
  • the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
  • TIL tumor infiltrating lymphocyte
  • the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
  • the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
  • the modified lymphocyte is a TCRy5+ lymphocyte.
  • the modified lymphocyte is a TCRy5+ lymphocyte and is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR.
  • the increased level of the immunosuppressive resistance gene LTBR results in increased killing of antigen-low cancer cells in tumor killing assays compared to an unmodified lymphocyte.
  • the tumor killing assay is an in vitro tumor killing assay.
  • the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
  • the modified lymphocyte is a regulatory T cell.
  • the modified lymphocyte is a human lymphocyte.
  • the modified lymphocyte is an autologous lymphocyte.
  • the modified lymphocyte comprises a vector comprising the expression cassette.
  • the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
  • the promoter is a ubiquitous promoter.
  • the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate-early enhancer and chicken betaactin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
  • the promoter is an inducible promoter.
  • the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
  • the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus. In some embodiments, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
  • a vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • a vector comprising nucleic acid encoding an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, and wherein the immunosuppressive resistance gene is selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIR
  • the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
  • the vector further comprises nucleic acid encoding a therapeutic protein.
  • the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the nucleic acid encoding the CAR or the TCR is included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
  • the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or the TCR is included in a second expression cassette.
  • the vector is a viral vector.
  • the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
  • vector is an episomal or non-integrating vector.
  • the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
  • the vector is a non-viral vector.
  • the non-viral vector is a plasmid.
  • the vector further comprises nucleic acid encoding a drugresistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
  • a modified lymphocyte comprising one or more of the vectors described herein.
  • composition comprising the modified lymphocyte as defined herein.
  • the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
  • provided herein is a method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the any vector described herein.
  • a method of increasing lymphocyte proliferation in an immunosuppressive cellular environment comprising introducing into lymphocytes the vector of any one of claims 44-56 or increasing expression of an immunosuppressive resistance gene selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC
  • an immunosuppressive resistance gene selected from the
  • the immunosuppressive cellular environment comprises a tumor microenvironment.
  • the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene is selected from the group consisting of COPZ2, ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, AD ARBI, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL
  • the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKD
  • the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, ADAT1, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A
  • the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene is selected from the group consisting of AB AT, ABHD12, ABH, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, ALOXE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B,
  • the immunosuppressive resistance gene is selected from the group consisting of CD47, CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZTBTB46.
  • a method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of: (i) collecting peripheral blood mononuclear cells (PBMCs) from an individual, (ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i), (iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and (iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
  • PBMCs peripheral blood mononuclear cells
  • the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR). In some embodiments, the exogenous nucleic acid further comprises a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the peripheral blood mononuclear cells are obtained from leukapheresis.
  • the CD8+ and CD4+ T cells are isolated sequentially.
  • the naive CD4+ T cells are differentiated into activated CD4+ T cells.
  • the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
  • the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
  • the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
  • the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
  • the transduced lymphocytes are enriched by positive selection.
  • the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
  • a method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte comprising: (i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual, (ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes, (iii) transiently stimulating the transduced lymphocytes, (iv) exposing the transduced lymphocytes to an immunosuppressive environment, (iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and (v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene.
  • the immunosuppressive cellular environment is selected from
  • FIGs. 1A-1B shows the dose-dependent suppression of T cell proliferation by Adenosine.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti- CD3/CD28 antibodies and cultured in the presence of increasing concentrations of adenosine for 4 days.
  • FIG. 1A quantifies the suppression by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of adenosine), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
  • FIG. 1A quantifies the suppression by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of adenosine), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
  • FIGs. 2A-2B shows the dose-dependent suppression of T cell proliferation by TGF-p.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of increasing concentrations of TGF-P for 4 days.
  • FIG. 2A quantifies the suppression by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of TGF-P), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
  • FIG. 2B shows the viability of T cells after 4 day culture in the presence of TGF-p.
  • FIGs. 3A-3E shows the dose-dependent suppression of T cell proliferation by coculture with nTregs and iTregs.
  • iTregs FIG. 3A
  • nTregs FIG. 3B
  • Grey histograms are isotype controls while blue histograms are cells stained with anti-FoxP3 antibody.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of increasing number of iTregs (FIG. 3C) or nTregs (FIG. 3D) for 4 days.
  • FIG. 3E shows the ex vivo expansion of iTreg and nTreg cells after isolation.
  • FIGs. 4A-4B displays the suppressive effect of polarized macrophages on T cell proliferation.
  • FIG. 4A shows representative staining of markers of immunosuppressive macrophage polarization.
  • FIG. 4B depicts the percent suppression of T cells cultured in the presence of macrophages.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of equal numbers of macrophages, polarized for 4 days with different combinations of the following factors: dexamethasone, IL-4, IL-6, TGF-
  • Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of macrophages), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
  • FIGs. 5A-5B shows the correlation of the percentage of proliferating T cells with their proliferation index (FIG. 5A) and absolute cell count (FIG. 5B). Gating on the most proliferating cells (the cells with highest dilution of CTY) correlates well with gold standard measures of T cell proliferation.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured with or without immunosuppression for 4 days.
  • Proliferation index the total number of divisions divided by the number of cells that went into division, was determined based on CTY dilution. Absolute cell count was measured by inclusion of absolute counting beads in the assay.
  • FIG. 6 shows a schematic representation of the immunosuppressive resistance gene screen in the context of TME immunosuppression.
  • FIGs. 7A-7B shows the effect of T cell stimulation on the ability to suppress T cells ex vivo.
  • Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti- CD3/CD28 antibodies and cultured in the presence of different immunosuppressive factors and cell types. Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of immunosuppression), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
  • the activator anti-CD3/CD28 antibodies
  • FIG. 7A shows the effect of T cell stimulation on the ability to suppress T cells ex vivo.
  • FIG. 7B shows the percent suppression of T cell proliferation obtained using a 400 lU/mL or 50 lU/mL concentration of IL-2 during the 4-day duration of the assay.
  • Nature 2022 indicates the methods used in the Legut et al., 2022 publication.
  • FIGs. 8A-8B depicts the quantification of screen quality metrics including coverage and cell number following T cell expansion (MM). Modifications to the immunosuppressive resistance gene screen enable multiple high quality immunosuppressive resistance gene screens from the same donor.
  • FIG. 8A depicts the average number of transduced T cells per barcode in the immunosuppressive resistance gene library.
  • FIG. 8B shows the total number of viable T cells after a 14-day expansion. Nature 2022 indicates the data published in Legut et al., 2022 publication.
  • FIG. 9 depicts the level of immunosuppression achieved in the context of the immunosuppressive resistance gene screens.
  • FIG. 10 shows the quantification of barcode recovery in each immunosuppressive resistance gene screen. Modifications to the immunosuppressive resistance gene screen enable improved data quality. Following sorting of the most proliferating T cells, the presence and relative abundance of each barcode present in the library was quantified by sequencing. Nature 2022 indicates the data published in Legut et al., 2022 publication.
  • FIG. 11 shows that overexpression of representative immunosuppressive resistance genes enhances CAR-T cell killing in the presence of regulatory T cell (Treg) immunosuppression in vitro.
  • CD4+ and CD8+ T cells co-expressing a HER-2- specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
  • FIG. 12 shows that overexpression of representative immunosuppressive resistance genes enhance CAR-T cell killing in the presence of adenosine immunosuppression in vitro.
  • CD4+ and CD8+ T cells co-expressing a HER2-specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
  • FIG. 13 shows that overexpression of representative immunosuppressive resistance genes enhance CAR-T cell killing in the presence of macrophage (mac) immunosuppression in vitro.
  • CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2- 28z CAR) and an indicated immunosuppressive resistance gene were co-incubated with GFP- engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
  • FIGs. 14A-14B show that overexpression of the immunosuppressive resistance gene ZBTB46 enhances CAR T cell killing of cancer cells in the presence of immunosuppression and alleviates T cell exhaustion in vitro.
  • CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and ZBTB46 or a control irrelevant gene were co- incubated with GFP-engineered HER2+ cancer cell lines at 1:4 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to account for the presence of a specified immunosuppressive factor on cancer cell growth. Mean and SEM are shown.
  • CAR T cells were co-incubated with cancer cells and were challenged with fresh cancer cells every 3-4 days. The assay was conducted in culture medium without (FIG. 14A) or with exogenous TGF-p (FIG. 14B).
  • FIG. 15 shows that overexpression of the immunosuppressive resistance gene LTBR results in alleviation of CAR-T cell exhaustion and enhanced cancer cell killing capacity in a repeated cancer challenge assay compared to benchmark CAR-T armoring genes (cJUN, mblL15, and TGFBR2dn) in vitro.
  • CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and LTBR were co-incubated with GFP-engineered HER2+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. When CAR T cells stopped specifically killing cancer cells, they were removed from the subsequent rounds of cancer cell challenge. Mean and SEM are shown.
  • FIG. 16 shows that overexpression of the immunosuppressive resistance gene LTBR, but not the benchmark CAR T armoring genes, enhances cancer cell killing in the presence of immunosuppressive factors in vitro.
  • CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and indicated gene were co-incubated with autologous regulatory T cells (Treg) and GFP-engineered HER2+ cancer cell lines at 1 : 1 T cells to cancer cells ratio. After 4 days, T cells were challenged with fresh cancer cells as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
  • FIG. 17 shows that overexpression of the immunosuppressive resistance gene LTBR enhances CAR T cell killing of CLDN6+ expressing ovarian cancer cell lines PAI and OV90 in vitro.
  • CD4+ and CD8+ T cells (aP T cells) co-expressing a CLDN6-specific CAR (CLDN6-BBz CAR) and LTBR or a control irrelevant gene were co-incubated with GFP- engineered CLDN6+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells, as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
  • FIGs. 18A-18C show that overexpression of the immunosuppressive resistance gene LTBR enhances cancer killing and T cells expansion while reducing T cell exhaustion of CAR gamma delta T cells in vitro.
  • Gamma delta T cells co-expressing a CLDN6-specific CAR (CLDN6-BBz CAR) and LTBR or a control irrelevant gene were co-incubated with 1 GFP-engineered CLDN6+ cancer cell line OV90. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. After 96 h of coincubation, T cells were harvested and stained in the presence of counting beads to determine their expansion and exhaustion. Mean and SEM are shown.
  • FIG. 18A shows percent of OV90 killing by CAR-T cells at 72 h of co-incubation at 1:32 T cell to cancer cell ratio.
  • FIG. 18B shows the fold-expansion of CAR gamma delta T cells with or without LTBR overexpression following co-incubation with OV90 cancer cells.
  • FIG. 18C analyzes the percentage of exhausted phenotypic CAR gamma delta T cells following co-incubation with OV90 cancer cells. Exhausted cells are defined as LAG3+ TIM3+.
  • FIG. 19 shows that overexpression of the immunosuppressive resistance gene LTBR enhances killing of antigen-low cancer cell line by CAR gamma delta T cells in vitro.
  • Gamma delta T cells co-expressing a CLDN6-specific CAR (CLDN6-28z CAR) and LTBR or a control irrelevant gene were co-incubated with GFP-engineered CLDN6 low cancer cell line SKOV3 for 88 h at 1:4 T cell to cancer cell ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
  • FIGs. 20A-20D show that LTBR enhances CLDN6-28z CAR efficacy in an animal model of ovarian cancer without causing apparent toxicities.
  • FIG. 20B shows body weight measurement since T cell injection, normalized to starting body weight. Dotted line represents a humane endpoint of 20% body weight loss.
  • FIG. 20C shows an assessment of tumor infiltration by human T cells at the experimental endpoint. At the experimental endpoint, matched tumor samples were explanted and processed to assess tumor infiltration by human T cells. Data normalized to the number of T cells detected in the tumor explanted from mice treated with CAR + tEGFR.
  • FIG. 21 shows enhanced CLDN6-28z y5 CAR efficacy with LTBR expression in a repeated challenge model in vitro.
  • CAR T cells, co-expressing a control gene tNGFR (“Unmodified CAR”) or armoring genes LTBR, mbIL15 or TGFpRIIDN were co-incubated with GFP+ OV90 ovarian cancer cells at 1:1 effector:target ratio for 72-96 hours. After each round of co-incubation, T cells were harvested and added to fresh cancer cells. Killing was determined at endpoint of each co-incubation by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone.
  • FIG. 22 shows enhanced CLDN6-28z aP CAR efficacy with LTBR expression in a tumor cell killing assay in vitro.
  • CAR T cells co-expressing a control gene tEGFR (“Unmodified CAR”), armoring gene LTBR or a truncated version of LTBR lacking the intracellular signaling domain (“LTBR del”) were co-incubated with GFP+ OV90 ovarian cancer cells at 1:32 effector: target ratio for up to 110 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone.
  • FIG. 23 shows enhanced CLDN6 « CAR T cell efficacy with LTBR expression in a tumor cell killing assay in vitro.
  • CAR T cells co-expressing a control gene tEGFR (“Unmodified CAR”) or armoring gene LTBR were co-incubated with GFP+ SKOV3 ovarian cancer cells at 1:16 effector: target ratio for up to 140 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991): Qhtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolim et af., Mol. Cell. Probes 8:91-98 (1994)).
  • the terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.
  • Nucleic acids described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins).
  • nucleic acid sequences encoding aspects of a CRISPR-Cas editing system described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject.
  • the genetic element is a vector.
  • the genetic element is a plasmid.
  • engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
  • ‘Variants” of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g., its specific inhibitory property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e., nonmutated physiological, sequence. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions.
  • amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bonds e.g., side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g., using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modem Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam). A variant may also include a non-natural amino acid.
  • a “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75, 100 or more amino acids of such protein or peptide, or over the full length of the protein or peptide.
  • gene can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • coding region and “region encoding” and grammatical variants thereof, refer to an open reading frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.
  • ORF open reading frame
  • Polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • nucleic acid sequence encoding an amino acid sequence includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • a nucleic acid sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • RNA Ribonucleic acid
  • protein Ribonucleic acid
  • expression may be transient or may be stable.
  • the terms “expressing”, and “overexpression” refer to increasing the expression of a gene or protein.
  • the terms refer to an increase in expression, for example, an increase in the amount of mRNA or protein expressed in a T cell, other lymphocyte or host cell, of at least 10%, as compared to a reference control level, or an increase of least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300% or at least about 400%.
  • the reference control level is the amount of mRNA or protein expressed in a cell that has not been transduced with nucleic acid encoding the protein.
  • Various methods for expression and/or overexpression are known to those of skill in the art, and include, but are not limited to, stably or transiently introducing a heterologous polynucleotide encoding a protein (i.e., an immunosuppressive resistance gene set forth in Tables 1-6) to be expressed and/or overexpressed in the cell or inducing expression or overexpression of an endogenous gene encoding the protein in the cell. It is understood that one or more genes set forth in Tables 1-6 can be expressed and/or overexpressed in a cell. It is also understood that two or more genes to be expressed and/or overexpressed in a cell can be selected from one or more of the genes set forth in Tables 1-6.
  • autologous refer to any material derived from the same subject to whom it is later to be re-introduced.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue, or system.
  • an exogenous nucleic acid includes an additional copy of a nucleic acid sequence already existing in the organism, cell, tissue, or system.
  • an exogenous nucleic acid includes vectors comprising nucleic acid encoding a gene that is already present at its endogenous location in the cell.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
  • an “expression cassette” refers to a nucleic acid molecule which encodes one or more ORFs or genes, e.g., an immunosuppressive resistance gene, or a CAR or TCR or component thereof.
  • An expression cassette also contains a promoter and may contain additional regulatory elements that control expression of one or more elements of a gene editing system in a host cell.
  • the expression cassette may be packaged into the capsid of a viral vector (e.g., a viral particle).
  • a viral vector e.g., a viral particle
  • such an expression cassette for generating a viral vector as described herein is flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • regulatory element or “regulatory sequence” refers to expression control sequences which are contiguous with the nucleic acid sequence of interest and expression control sequences that act in trans or at a distance to control the nucleic acid sequence of interest.
  • regulatory elements comprise but are not limited to: promoter; enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and poly adenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Also, see Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleic acid sequence in many types of target cell and those which direct expression of the nucleic acid sequence only in certain target cells (e.g., tissue-specific regulatory sequences).
  • a “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • the term “constitutive” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • the term “inducible” or “regulatable” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • the inducible promoter is activated in response to T cell stimulation.
  • the promoter is heterologous. In some embodiments, the promoter is an NF AT, API, NFKB, or IRF4 promoter.
  • tissue- specific when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • Exemplary promoters include the CMV IE gene, EF-la., ubiquitin C, 5’LTR, or phosphoglycerokinase (PGK) promoters.
  • operably linked refers to functional linkage between one or more regulatory sequences and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein coding regions, are in the same reading frame.
  • lentivirus refers to a genus of the Retroviridae family. Eentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • one or more genes are encoded by a nucleic acid sequence that is delivered to a host cell by a vector or a viral vector, of which many are known and available in the art.
  • a vector comprising an expression cassette as described herein.
  • a vector is a non- viral vector.
  • a vector is a viral vector.
  • a “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of interest is packaged in a viral capsid or envelope.
  • viral vectors include but are not limited to lentivirus, adenoviruses, retroviruses (y- retroviruses and lentiviruses), poxviruses, adeno- associated viruses (AAVs), baculoviruses, herpes simplex viruses.
  • the viral vector is replication defective.
  • a “replication-defective virus” refers to a viral vector, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient, i.e., they cannot generate progeny virions but retain the ability to infect cells.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • the vector is a non-viral plasmid that comprises an expression cassette described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.
  • an expression cassette described herein e.g., naked DNA, naked plasmid DNA, RNA, and mRNA
  • various compositions and nano particles including, e.g., micelles, liposome
  • an expression cassette as described herein is engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for introduction to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon.
  • a suitable genetic element e.g., naked DNA, phage, transposon, cosmid, episome, etc.
  • the selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • RNA or DNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-11 (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the modified lymphocytes disclosed herein display significantly improved proliferation when exposed to immunosuppressive cellular environments mimicking an immunosuppressive tumor microenvironment (TME).
  • TEE immunosuppressive tumor microenvironment
  • the immunosuppressive resistance genes disclosed herein comprise both immune response modifiers as well as genes not typically expressed by peripheral T-cells.
  • the modified lymphocytes expressing the immunosuppressive resistance genes disclosed herein therefore address an important limitation currently impeding the establishment of effective cellular therapies for tumors comprising immunosuppressive microenvironments .
  • modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the immunosuppressive resistance gene when expressed in a lymphocyte, enables the lymphocyte to proliferate despite exposure to an immunosuppressive cellular environment.
  • the immunosuppressive cellular environment is selected from selected from the group consisting of adenosine driven immunosuppression, TGF-P driven immunosuppression, regulatory T cell (Treg) driven immunosuppression, and macrophage driven immunosuppression.
  • the immunosuppressive mechanism underlying adenosine driven immunosuppression comprises the adenosinergic pathway.
  • activation of the adenosinergic pathway occurs within hypoxic tumors.
  • the adenosinergic pathway comprises ectonucleotidases (CD39 and CD73) and adenosine receptors (AIR, A2AR, A2BR and A3R) that participate in the generation and signalling of adenosine in the tumour microenvironment (TME).
  • TME tumour microenvironment
  • the cyclic AMP (cAMP)-activating receptors A2AR and A2BR predominantly exert immunosuppressive functions in the TME.
  • extracellular adenosine exerts local suppression through tumor-intrinsic and host-mediated mechanisms.
  • the mechanism underlying TGF-P driven immunosuppression comprises inhibition of T cell proliferation.
  • exposure to TGF-P inhibits the IL-2 expression and secretion in T cells.
  • exposure to TGF- P inhibits expression of the Ifiig, Gzma, Gzmb, Prfl and Faslg genes in T cells.
  • exposure to TGF-P downregulates the expression of MHC molecules on the surface of tumor cells.
  • Treg cells directly suppress anticancer immunity, thereby hampering effective anti-tumor immune responses in tumor-bearing individuals.
  • Tregs alter immune function in cells of both the innate and adaptive immune systems.
  • Tregs secrete immunosuppressive cytokines such as TGF-P, IL-10, and IL-35.
  • Tregs inhibit the metabolic functions of immune cells through CD25 (IL-2 receptor alpha) dependent cytokine deprivation facilitated apoptosis, immunosuppressive adenosine by ectoenzymes CD39 and CD73 and c-AMP mediated inhibition.
  • the mechanism underlying macrophage driven immunosuppression comprises the activity of Tumor-Associated Macrophages (TAMs).
  • TAMs express inhibitory cytokines that dampen immune responses.
  • macrophages produce oxygen radicals that generate toxic compounds, including peroxynitrite and hydrogen peroxide, that suppress the proliferation and activity of immune cells.
  • macrophages directly suppress T cell activity and proliferation.
  • modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene set forth in Tables 1- 6.
  • the immunosuppressive resistance gene is endogenously expressed by the lymphocyte.
  • the immunosuppressive resistance gene is not naturally expressed by the lymphocyte.
  • the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by at least about 10% more as compared to the amount of mRNA of the immunosuppressive resistance gene expressed in a non-modified lymphocyte.
  • the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by between about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% about 90% to about 100% more as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte. In some embodiments, the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by at least about 1.2-fold as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte.
  • the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by about 1.2-fold, about 1.5-fold, about 1.7-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, or about 10-fold as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte.
  • this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 1, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking an adenosine- driven tumor microenvironment compared to an unmodified lymphocyte.
  • the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, AD ARBI, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP
  • this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 2, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a TGF-P- driven tumor microenvironment compared to an unmodified lymphocyte.
  • the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, C
  • this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 3, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a macrophage- driven tumor microenvironment compared to an unmodified lymphocyte.
  • the immunosuppressive resistance gene is selected from the group consisting of ABAT, ABHD12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, AL0XE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GALNT2, B3GALT4, B3
  • this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 4, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a regulatory T cell-driven tumor microenvironment compared to an unmodified lymphocyte.
  • the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, ADAT1, ADGRE5, ADIPOR2, AD0RA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, A0C1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1,
  • the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46
  • the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MYO1A, MYOC, MYOM3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
  • modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the therapeutic protein comprises a chimeric antigen receptor (CAR).
  • the therapeutic protein comprises a T cell receptor (TCR).
  • chimeric antigen receptor or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
  • the stimulatory molecule is TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4- IBB, or CD3-zeta.
  • the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the stimulatory molecule is 4-IBB. In some embodiments, the stimulatory molecule is CD28. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below (also referred to as a “co stimulatory signaling domain”).
  • the costimulatory molecule is chosen from a costimulatory molecule described herein, e.g., 0X40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD1 la/CD18), ICOS and 4-IBB (CD 137), or any combination thereof.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule (a primary signaling domain).
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule (a costimulatory signaling domain) and a functional signaling domain derived from a stimulatory molecule (a primary signaling domain).
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the scFv domain during cellular processing and localization of the CAR to the cellular membrane.
  • the extracellular domain of the CAR binds to a tumor antigen.
  • the tumor antigen is HER2.
  • the tumor antigen is Claudin-6 (CLDN6).
  • the tumor antigen is expressed at a low level in a cancer cell or tumor cell.
  • the cancer cell or the tumor cell is an antigen-low cancer cell or an antigen-low tumor cell.
  • the CAR binds to CLDN6.
  • the CAR is a CLDN6-CAR.
  • the CLDN6-CAR can comprise any one of the nucleotide and/or amino acid sequences described in US 2022/0306711 Al, US 2022/0339193 Al, US 10,561,686 B2, and Mackensen et al., Nat. Med., 2023, 29:2844-2853, all of which are herein incorporated by reference in their entireties.
  • the modified lymphocyte comprises a nucleic acid sequence encoding a CAR and further comprises a nucleic acid sequence encoding an immunosuppressive resistance gene described herein.
  • TCRs T Cell Receptors
  • the modified lymphocyte expresses a TCR.
  • the TCR is a disulfide-linked membrane- anchored heterodimer present on T cell lymphocytes, and the majority of T cells are aP T cells having a TCR consisting of an alpha (a) chain and a beta (P) chain.
  • each chain comprises a variable (V) and a constant (C) domain, wherein the variable domain recognizes an antigen, or an MHC -presented peptide.
  • TCRa and TCRP chains with a known specificity or affinity for specific antigens, e.g., tumor antigens described herein, can be introduced to a T cell using the methods described herein.
  • TCRa and TCRP chains having increased specificity or affinity for a particular antigen can be isolated using standard molecular cloning techniques known in the art.
  • other modifications that increase specificity, avidity, or function of the TCRs or the engineered T cells expressing the TCRs can be readily envisioned by the ordinarily skilled artisan, e.g., promoter selection for regulated expression, mutations in the antigen binding regions of the TCRa and TCRP chains.
  • Any isolated or modified TCRa and TCRP chain can be operably linked to or can associate with one or more intracellular signaling domains described herein.
  • signaling can be mediated through interaction between the antigen-bound ab heterodimer to CD3 chain molecules, e.g., CD3zeta (z).
  • a smaller subset of T cells expresses a TCR having a (y) gamma chain and a delta (6) chain.
  • gamma-delta (y 6) T cells make up 3-10% of circulating lymphocytes in humans, and the Vd2+ subset can account for up to 95% of y6 T cells in blood.
  • V62+ cells recognize non-peptide epitopes and do not require antigen presentation by major histocompatibility complexes (“MHC”) or human leukocyte antigen (“HLA”).
  • MHC major histocompatibility complexes
  • HLA human leukocyte antigen
  • V62+ T cells also express a Vy9 chain and are stimulated by exposure to 5-carbon pyrophosphate compounds that are intermediates in mevalonate and non-mevalonate sterol/isoprenoid synthesis pathways.
  • the response to isopentenyl pyrophosphate (5-carbon) is universal among healthy human beings.
  • another subset of y6 T cells, V61+ make up a much smaller percentage of the T cells circulating in the blood, but are commonly found in the epithelial mucosa and the skin.
  • y6 T cells have several functions, including killing tumor cells and pathogen-infected cells.
  • stimulation through the y6 TCR improves the capacity for cellular cytotoxicity, cytokine secretion and other effector functions.
  • the TCRs of y6 T cells have unique specificities and the cells themselves occur in high clonal frequencies, thus allowing rapid innate-like responses to tumors and pathogens. See, e.g., Park and Lee, Exp Mol Med. 2021 Mar;53(3):318-327., which is incorporated herein by reference.
  • the modified lymphocyte comprises a nucleic acid sequence encoding a TCR and further comprises a nucleic acid sequence encoding an immunosuppressive resistance gene described herein.
  • modified lymphocytes comprising exogenous nucleic acids encoding an immunosuppressive resistance gene described herein.
  • the modified lymphocyte comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene.
  • the modified lymphocyte comprises an expression cassette comprising nucleic acids encoding one or more immunosuppressive resistance genes.
  • the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in the same vector. In some embodiments, the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in the same expression cassette. In some embodiments, the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in different vectors.
  • the immunosuppressive resistance gene comprises any of the genes identified in Tables 1-6. As used herein, where reference to a specific gene of Tables 1- 6 is mentioned, it is intended that the use of the coding sequence for the full-length protein, a fragment having a deletion or truncation, or a variant having one or more substitutions in the amino acid, is intended.
  • the nucleic acid encodes a full-length protein. In some embodiments, the nucleic acid encodes a functional fragment of a truncated protein. In some embodiments, the functional fragment of the truncated protein comprises one or more functional domains of the protein.
  • the functional fragment of the truncated protein may comprise catalytic activity. In some embodiments, the functional fragment of the truncated protein may facilitate protein-protein, protein-DNA, or protein- RNA interactions.
  • the immunosuppressive resistance gene encodes a variant protein having one or more substitutions in the amino acid.
  • the nucleic acid encodes a protein sequence having a deletion or truncation in the N terminus. In some embodiments, the nucleic acid encodes a protein sequence having a deletion or truncation in the C terminus.
  • the nucleic acid encodes a protein having of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.
  • the expression cassette includes more than one effectorenhancing gene, or functional fragment or variant thereof. In some embodiments, the expression cassette encodes at least two immunosuppressive resistance genes. In some embodiments, the expression cassette encodes one, two, three, four, or five immunosuppressive resistance genes. In some embodiments, the expression cassette encodes between about one and about five immunosuppression resistance genes.
  • the modified lymphocyte comprises two or more exogenous nucleic acids encoding two or more of the immunosuppressive resistance genes described herein. In some embodiments, each immunosuppressive resistance gene is encoded in a separate expression cassette.
  • the modified lymphocyte comprises a first expression cassette encoding a first immunosuppression resistance gene, and a second expression cassette encoding a second immunosuppressive resistance gene. In some embodiments, the modified lymphocyte comprises two, three four, or five expression cassettes, wherein each expression cassette encodes a unique immunosuppression resistance gene.
  • the expression cassette comprises a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
  • the promoter is heterologous.
  • the promoter is a ubiquitous promoter.
  • the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate-early enhancer and chicken betaactin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
  • the promoter is an inducible promoter.
  • the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
  • the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte. In some embodiments, the expression cassette comprising the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus. In some embodiments, the safe harbor locus is selected from the group consisting of AAVS1, hROSA26, and CCR5. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
  • the expression cassette further comprises a therapeutic protein.
  • the therapeutic protein comprises a CAR or a TCR.
  • vectors comprising the nucleic acid encoding an immunosuppressive resistance gene set forth in any one of Tables 1-6.
  • the nucleic acid encodes a full-length protein or a functional fragment or variant thereof.
  • the vector comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene.
  • the vector further comprises a nucleic acid encoding a therapeutic protein.
  • the therapeutic protein comprises a CAR or a TCR.
  • the vector comprises an expression cassette comprising the nucleic acid encoding the CAR or TCR.
  • the vector comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR. In some embodiments, the vector comprises a first expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene and a second expression cassette comprising the nucleic acid encoding the CAR or TCR.
  • the vector further comprises nucleic acid encoding a drugresistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
  • the drug-resistance gene is selected from the group consisting of neomycin resistance gene (NeoR), kanamycin resistance gene (NPTII), puromycin-N-acetyl Transferase (PAC), aminoglycoside phosphotransferase (APH), and blasticidin S deaminase (BSD).
  • the drug-resistance gene is a puromycin-N-acetyltransferase (PAC) gene.
  • the surface expressed safety switch gene is selected from the group consisting of CD20, HER2, EGFR, full length NGFR, and truncated NGFR.
  • the vector is a viral vector.
  • the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
  • the vector is an episomal or non-integrating vector.
  • the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
  • the vector is a non-viral vector.
  • the non-viral vector is a plasmid.
  • the non-viral vector is delivered to a lymphocyte by electroporation or cell squeezing.
  • the non-viral vector is encapsulated in nanoparticles or liposomes.
  • modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • the therapeutic protein is a CAR or a TCR.
  • the CAR or the TCR binds to a tumor antigen.
  • the tumor antigen is HER2 or Claudin-6.
  • the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR.
  • the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, FOSB, GSDME, IL26, LIMA1, LTBR, PTK6, SKIL, SRC, STK11, and YBX2.
  • the modified lymphocyte comprises a CAR that binds to HER2, and the immunosuppressive resistance gene ZBTB46.
  • the modified lymphocyte comprises a CAR that binds to HER2, and the immunosuppressive resistance gene LTBR.
  • the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of LTBR, cJUN, mbIL15, and TGFBR2dn. In some embodiments, the modified lymphocyte comprises a CAR that binds to CLDN6 and the immunosuppressive resistance gene LTBR.
  • the modified lymphocyte comprises a mutant allele of the immunosuppressive resistance gene LTBR.
  • the mutant allele encodes for a non-functional LTBR.
  • the non-functional LTBR is LTBR-del.
  • LTBR-del is a non-functional, truncated version of LTBR lacking the intracellular signaling domain.
  • lack of an intracellular signaling domain results in the inability of LTBR to transmit signaling even when the extracellular part binds a ligand.
  • the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
  • the modified lymphocyte is a CD3 + lymphocyte. In some embodiments, the modified lymphocyte is a TCRaP + CD4" CD8" T cell. In some embodiments, the modified lymphocyte is a CD4 + T cell or a CD8 + T cell. In some embodiments, the modified lymphocyte is a CD4 + T cell. In some embodiments, the modified lymphocyte is a CD8 + T cell. In some embodiments, the CD4 + T cell or a CD8 + T cell expresses an aP TCR. In some embodiments, the modified lymphocyte is a y5 T cell. In some embodiments, the y5 T cell expresses a y5 TCR. In some embodiments, the modified lymphocyte is a human lymphocyte.
  • the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
  • the modified lymphocyte is a regulatory T cell.
  • the modified lymphocyte is derived from a cell in culture. In some embodiments, the modified lymphocyte is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is engineered to knock out or silence the expression of TCR and HLA proteins. In some embodiments, the iPSC is further engineered to express immune receptors, cytokines, chemokines, or other immune regulatory factors to enhance anti-tumor function.
  • iPSC induced pluripotent stem cell
  • the modified lymphocyte is derived from a lymphocyte isolated from an individual. In some embodiments, the modified lymphocyte is derived from a lymphocyte isolated from a blood sample obtained from an individual. In some embodiments, the modified lymphocyte is derived from a lymphocyte isolated from a tumor sample obtained from an individual. In some embodiments, the modified lymphocyte is derived from a tumor infiltrating lymphocyte (TIL). In some embodiments, the modified lymphocyte is a tumor infiltrating lymphocyte (TIL). In some embodiments, the modified lymphocyte is a cell therapy.
  • TIL tumor infiltrating lymphocyte
  • TIL tumor infiltrating lymphocyte
  • compositions Comprising Modified Lymphocytes
  • a pharmaceutical composition comprising modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene set forth in any one of Tables 1-6.
  • cells in the composition have anti-tumor activity.
  • the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • the pharmaceutical composition comprises a modified lymphocyte comprising nucleic acid encoding an immunosuppressive resistance gene and further comprises a nucleic acid encoding a therapeutic protein.
  • the therapeutic protein comprises a CAR or TCR.
  • the pharmaceutical composition comprises a modified T cell, a modified NK cell, or a modified NK T cell.
  • the pharmaceutical composition comprises a modified CD3+ lymphocyte. In some embodiments, the pharmaceutical composition comprises a modified TCRaP+ CD4- CD8- T cell. In some embodiments, the pharmaceutical composition comprises a modified CD4+ T cell or a modified CD8+ T cell. In some embodiments, the pharmaceutical composition comprises a modified y5 T cell. In some embodiments, the pharmaceutical composition comprises a modified human lymphocyte.
  • the composition is in the form of a liquid.
  • the liquid is a cellular suspension.
  • the liquid is useful for delivery by injection.
  • the liquid comprises a cell culture media.
  • a composition for administration by injection in addition to the modified lymphocytes, contains one or more excipients selected from the group consisting of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent.
  • kits for increasing lymphocyte proliferation in an immunosuppressive cellular environment comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 1-6.
  • the immunosuppressive cellular environment comprises a tumor microenvironment (TME).
  • TME tumor microenvironment
  • the immunosuppressive cellular environment recapitulates the in vivo immunosuppressive cellular environment within the TME.
  • the immunosuppressive cellular environment is an in vitro immunosuppressive cellular environment.
  • the immunosuppressive cellular environment established in and around tumors comprises adenosine driven immunosuppression, TGF-P driven immunosuppression, regulatory T cell (Treg) driven immunosuppression, and/or macrophage driven immunosuppression.
  • the method comprises modifying a population of human lymphocytes with a lentiviral vector comprising an expression cassette comprising nucleic acids encoding an immunosuppressive resistance gene set forth in Tables 1-6, and optionally, a CAR or TCR.
  • the modified lymphocytes described herein are expanded.
  • the modified lymphocytes are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days).
  • the method comprises increasing lymphocyte proliferation in an adenosine driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene conferring increased lymphocyte proliferation in an adenosine driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 1.
  • the method comprises increasing lymphocyte proliferation in a TGF-P driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a TGF- P driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 2.
  • the method comprises increasing lymphocyte proliferation in a Treg driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a Treg driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 3.
  • the method comprises increasing lymphocyte proliferation in a macrophage driven immunosuppressive cellular environment.
  • the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a macrophage driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 4.
  • the method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene described herein comprises the steps of: (i) collecting peripheral blood mononuclear cells (PBMCs) from an individual, (ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cell from the PBMCs of step (i), (iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and (iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
  • PBMCs peripheral blood mononuclear cells
  • the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR). In some embodiments, the exogenous nucleic acid further comprises a T cell receptor (TCR).
  • the peripheral blood mononuclear cells are obtained from leukapheresis. In some embodiments, the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
  • the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element. In some embodiments, the transduced lymphocytes are enriched by positive selection.
  • the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
  • the CD8+ and CD4+ T cells are isolated sequentially.
  • the naive CD4+ T cells are differentiated into activated CD4+ T cells.
  • the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
  • the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
  • identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte comprising: (i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual, (ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes, (iii) transiently stimulating the transduced lymphocytes, (iv) exposing the transduced lymphocytes to an immunosuppressive environment, (iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and (v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene.
  • the immunosuppressive cellular environment is selected from the group consist
  • the method comprises a genome-scale gain-of-function screen in primary human CD4+ and CD8+ T-cells.
  • the lymphocyte population comprising a mixture of CD4+ and CD8+ cells is obtained from a human blood sample.
  • the mixture of CD4+ and CD8+ cells is obtained from an enriched apheresis product.
  • the enriched apheresis product comprises peripheral blood mononuclear cells (PBMCs).
  • the human blood sample is obtained from a healthy blood donor.
  • the CD8+ and CD4+ T cells are isolated from the PBMCs sequentially. In some embodiments, the PBMCs are not pooled together prior to the isolation of the CD8+ and CD4+ T cells.
  • the CD8+ and CD4+ T cells are isolated from the PBMCs sequentially.
  • isolating the CD8+ and CD4+ T cells comprises a first step of isolating CD8+ T cells from the PBMCs using magnetic selection of the CD8 protein, followed by a second step of isolating the CD4+ T cells from the PBMCs using magnetic selection of the CD4 protein.
  • the method further comprises isolating monocytes from the PBMCs following the isolation of CD8+ and CD4+ T cells from the PBMCs.
  • the method further comprises isolating naive CD4+ T cells from the PBMCs using magnetic selection of the CD3 protein, the CD4 protein, and the CD45RA protein.
  • the isolated mixture of CD4+ and CD8+ cells are cultured in T cell media supplemented with recombinant human IL-2.
  • the CD4+ and CD8+ T cells are activated by culturing the CD4+ and CD8+ T cells with antibody complexes that bind to and cross-link CD3 and CD28 surface ligands on the CD4+ and CD8+ T cells.
  • the CD4+ and CD8+ T cells are transduced with a plurality of viral vectors, wherein the viral vectors are lentiviral vectors. In some embodiments, the CD4+ and CD8+ T cells are transduced with the plurality of viral vectors about 24 hours following isolation from the PBMCs. In some embodiments, the CD4+ and CD8+ T cells are transduced with the plurality of viral vectors between about 20 hours to 22 hours, between about 22 hours to about 24 hours, or between about 24 hours to about 26 hours following isolation from the PBMCs.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the CD4+ and CD8+ T cell population is transduced with the viral vector that delivers genes to be screened.
  • the viral vector comprises an expression cassette comprising a promoter operably linked to a barcoded gene.
  • the viral vector comprises a lentiviral-mediated delivery of an open reading frame (ORF) library (see Sack et al. Cell. 2018 Apr 5;173(2):499-514.e2, which is incorporated herein by reference).
  • ORF open reading frame
  • barcode or “barcode sequence” as used herein refers to a nucleotide sequence that corresponds to and allows for detection and/or identification of an expressed gene.
  • the barcode typically comprises four or more nucleotides.
  • the barcode comprises 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, or 15 nucleotides.
  • the barcode comprises 8 to 15 nucleotides.
  • the terms “barcoded gene”, “barcoded ORF”, and the like refers to a nucleic acid that has an appended barcoded sequence, whether the barcode is linked directly to the 5’ or 3’ end of the ORF or separated by 1 or more nucleotides at the 5’ or 3’ end of the ORF.
  • the transduced CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene.
  • the drug-resistance gene comprises the puromycin N-acetyltransferase gene.
  • the transduced CD4+ and CD8+ T cells are selected for the expression of puromycin N-acetyltransferase by exposing the CD4+ and CD8+ T cells to puromycin.
  • the CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene about 72 hours following isolation from the PBMCs.
  • the CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene between about 68 to about 70 hours, about 70 to about 72 hours, or about 72 hours to about 74 hours following isolation from the PBMCs.
  • the transduced CD4+ and CD8+ T cells were cultured in media supplemented with puromycin throughout the culture period.
  • the viral vector further comprises nucleic acid encoding a selection gene (e.g., a fluorescent protein, GFP) that facilitates the further isolation or enrichment of transduced CD4+ and CD8+ T cells using flow cytometry.
  • a selection gene e.g., a fluorescent protein, GFP
  • the CD4+ and CD8+ T cells were cultured according to standard cell culture techniques commonly known in the art.
  • the CD4+ and CD8+ T-cells were either split or had media replaced to maintain a cell density of between about IxlO 6 to about 2xl0 6 cells per mL about every 2 days.
  • the CD4+ and CD8+ T-cells were either split or had media replaced to maintain a cell density of between about IxlO 6 to about 2xl0 6 cells per mL about every 3 days.
  • a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to an immunosuppressive cellular environment.
  • the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
  • a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to an adenosine-driven immunosuppressive cellular environment.
  • the subset of transduced lymphocytes is cultured in the presence of between about 1 pM to about 64 pM of adenosine.
  • the subset of transduced lymphocytes is cultured in the presence of between about 1 pM to about 4 pM, between about 4 pM to about 16 pM, or between about 16 pM to about 64 pM of adenosine.
  • the subset of transduced lymphocytes is cultured in the presence of about 1 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 4 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 16 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 64 pM of adenosine.
  • a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a TGF-P-driven immunosuppressive cellular environment.
  • the subset of transduced lymphocytes is cultured in the presence of between about 0.25 pM to about 256 pM of TGF-p.
  • the subset of transduced lymphocytes is cultured in the presence of between about 0.25 pM to about 1 pM, between about 1 pM to about 4 pM, between about 4 pM to about 16 pM, between about 16 pM to about 64 pM, or between about 64 pM to about 256 pM of TGF-p.
  • the subset of transduced lymphocytes is cultured in the presence of about 0.25 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 1 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 4 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 16 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 64 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 256 pM of TGF-p.
  • a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a Treg-driven immunosuppressive cellular environment.
  • the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of between about 16:1 to about 1:1.
  • the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of between about 16:1 to about 8:1, about 8:1 to about 4:1, about 4:1 to about 2:1, or about 2:1 to about 1:1.
  • the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 16:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 8: 1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 4:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 2:1.
  • the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 1 : 1.
  • the Treg cells are differentiated induced Treg cells (iTreg).
  • the Treg cells are natural Treg cells (nTreg).
  • a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a macrophage-driven immunosuppressive cellular environment.
  • the subset of transduced lymphocytes is cultured in the presence of macrophage cells at a suppressor to effector ratio of about 1:1.
  • the macrophage cells are polarized macrophages.
  • identification of the expressed gene is determined by PCR amplification of the gene and/or barcode sequence.
  • PCR is performed on genomic DNA (gDNA) obtained from the lymphocytes.
  • a reverse transcription step is performed to generate cDNA form the cell transcriptome and/or from an exogenous gene and barcode mRNA transcript.
  • the amplified DNA products are then sequenced to identify an exogenous gene expressed in the isolated lymphocyte and/or to quantify the relative expression of an exogenous gene in a population of isolated lymphocytes.
  • the disclosed methods include RNA and/or DNA sequencing of lymphocytes using techniques that include, but are not limited to, whole transcriptome analysis, whole genome analysis, barcoded sequencing of whole or targeted regions of the genome, and combinations thereof.
  • RNA and/or DNA sequencing is performed in combination with proteome analysis.
  • the methods include detection of cell surface or intracellular proteins using, e.g., flow cytometry.
  • the methods comprise detection or identification the barcoded gene in combination with profiling additional molecular modalities using methods described in the art, including for example single-cell sequencing analysis (e.g., 10X Genomics Multiome platform), single-cell RNA-sequencing (scRNA-seq) (See, e.g., Haque et al. Genome Medicine, 9, Article number: 75 (2017); Hwang et al. Exp Mol Med. 2018 Aug 7;50(8):96), cell-hashing (See, e.g., Stoeckius et al. Genome Biol. 2018; 19: 224), Perturb-Seq. (See, e.g., Dixit et al. Cell.
  • single-cell sequencing analysis e.g., 10X Genomics Multiome platform
  • scRNA-seq single-cell RNA-sequencing
  • cell-hashing See, e.g., Stoeckius et al. Genome Biol. 2018; 19:
  • CITE-seq cellular indexing of transcriptomes and epitopes-seq
  • OverCITE-seq overexpression-compatible cellular indexing of transcriptomes and epitopes by sequencing
  • Example 1 Methods to improve T cell function in an immunosuppressive environment.
  • Leukopaks from de-identified healthy donors were collected by and purchased from StemcellPeripheral blood mononuclear cells (PBMC) were isolated from leukopaks using Lymphoprep (Stemcell) gradient centrifugation.
  • PBMC StemcellPeripheral blood mononuclear cells
  • CD8+ and CD4+ were isolated sequentially from the same donor.
  • CD8+ T-cells were isolated by magnetic positive selection using EasySep Human CD8 Positive Selection Kit II (Stemcell).
  • Stemcell EasySep Human CD8 Positive Selection Kit II
  • CD4+ T-cells were isolated from the resulting flowthrough by positive magnetic selection using EasySep Human CD4+ Positive Selection Kit II (Stemcell). The flowthrough from the CD4+ T cell isolation was used as a source of monocytes.
  • Naive CD4+ T cells were isolated from PBMCs by magnetic selection using EasySep Human Naive CD4+ T Cell Isolation Kit. Regulatory T cells (Treg) were isolated using EasySep Human CD4+CD1271owCD25+ Regulatory T Cell Isolation Kit.
  • naive CD4+ T cells, CD4+ or CD8+ T-cells were resuspended in T-cell Media, which consisted of Immunocult-XF T-cell Expansion Medium (Stemcell) supplemented with recombinant human IL-2 (Stemcell). Additionally, the media for naive CD4+ T cells was supplemented with TGF-
  • CD4+ or CD8+ T cells were transduced with concentrated lentivirus 24 h post isolation. 72 h post isolation, lentivirally transduced CD4+ or CD8+ T cells were selected with puromycin. Every 2-3 days CD4+ and CD8+ T-cells were either split or had media replaced to maintain cell density of 1-2 x 10 6 cells per mL. Lentivirally transduced CD4+ or CD8+ T cells were maintained in media containing puromycin for the duration of culture. T- cells were used for phenotypic or functional assays fresh or cryopreserved in Bambanker Cell Freezing Media (Bulldog Bio).
  • the flowthrough enriched in monocytes was plated to achieve -90% confluence after 10 days of culture in RPMI 1640 media supplemented with FBS and cytokines M-CSF and GM-CSF (Stemcell). From day 5 after isolation, the culture media was further supplemented with at least of the following factors: dexamethasone, IL-4, IL-6, TGF-
  • HEK293FT cells were obtained from Thermo Fisher and cultured in DMEM supplemented with 10% Serum Plus-II (Thermo Fisher).
  • OV90 cells were obtained from American type culture collection (ATCC) and were cultured per ATCC recommendations. Endogenous Claudin 6 expression was verified by standard flow cytometry techniques using commercially available antibodies.
  • Lentivirus encoding the pooled ORF library was produced by co-transfecting a third- generation lentiviral transfer plasmid pool (cite 2nd application) together with the packaging plasmid psPAX2 and envelope plasmid pMD2.G into HEK293FT cells, using polyethyleneimine linear MW 25000 (Poly sciences). After 72 hours, the supernatants were harvested and filtered through a 0.45 pm Steriflip-HV filter (Millipore). The virus was concentrated using Lentivirus Precipitation Solution (Alstem). The concentrated lentivirus was then resuspended in T-cell Media containing IL-2 and stored at -80°C.
  • the cells were stained with the specific antibody or isotype control for 30 minutes in the dark at room temperature. Finally, the cells were washed twice in the appropriate permeabilization buffer and acquired on a flow cytometer. Gating was performed using appropriate isotype, fluorescence minus one and biological controls. Typically, 5,000- 10,000 live events were recorded per sample.
  • CD4+ and CD8+ T-cells were isolated from PBMC from three healthy donors and cultured separately for the first 9 days.
  • the amount of lentivirus used for transduction was titrated to result in -40% transduction efficiency, to minimize the probability of multiple ORFs being introduced into a single cell.
  • the cells were maintained in T-cell media containing puromycin and counted every 2-3 days to maintain cell density of 1-2 x 10 6 cells/mL.
  • T-cells were harvested, counted, pooled at a 1:1 CD4:CD8 ratio and plated back in T cell media.
  • CTYlow and CTYhigh cells (corresponding to the bottom 20% and top 20% of the distribution) were sorted using Sony MA900 or BD FACSAria cell sorter. Genomic DNA was isolated, and two rounds of PCR to amplify ORF barcodes and add Illumina adaptors were performed as described previously (Legut, M. et al. Nature 603, 728-735 (2022)). Pooled ORF screen analysis
  • Barcodes were mapped to the reference library after adaptor trimming with cutadapt using bowtie. All subsequent analyses were performed in RStudio with R. To calculate individual barcode enrichment, barcode counts were normalized to the total number of reads per sample (with pseudocount added) and log2 transformed. Statistical analysis on barcode enrichment was performed using MAGeCK, comparing CTY 1OW samples to corresponding CTY hlgh samples for each immunosuppressive conditions, using three donors and two cell types (CD4+, CD8+) as replicates.
  • Example 2 In vitro assays to recapitulate immunosuppressive cellular environments and methods to quantify T cell proliferation.
  • mediators of TME immunosuppression comprise adenosine, TGF-P, regulatory T cells, and immunosuppressive macrophages.
  • the following example establishes methods to assess T cell proliferation following exposure to these mediators of TME immunosuppression.
  • T cells were cultured in the presence varying concentrations of either agent while monitoring their proliferation and viability.
  • CD4+ and CD8+ T cells experience a dose-dependent suppression of proliferation when cultured in the presence of adenosine.
  • this dose-dependent suppression of proliferation was not a result of a reduction in cell viability (FIG. IB).
  • a similar dose dependent inhibition of proliferation was observed when CD4+ and CD8+ T cells were cultured in the presence of TGF-P (FIG. 2A).
  • T cell proliferation in the presence of TGF-P was also not a result of a reduction in T cell viability (FIG. 2B).
  • Regulatory T cells were also identified as contributors to immunosuppressive environments.
  • natural Tregs nTregs
  • iTregs differentiated induced Tregs
  • Both nTregs and iTregs showed robust expression of the key transcription factor FoxP3, were able to suppress proliferation of CD4+ and CD8+ T cells in a dose-dependent manner and expanded well ex vivo (FIGs. 3A-3E).
  • polarized macrophages showed phenotypic hallmarks of immunosuppressive M2-like macrophages, such as a decreased expression of CD64 and an increased expression of CD206 and PD-L1 (FIG. 4A).
  • the polarized macrophages were also capable of suppressing T cell proliferation across a range of polarization conditions (dexamethasone, IL-4, IL-6, TGF-pi, TGF-P2, TGF-P3, IL-10, IL-ip) (FIG. 4B).
  • a proliferation assay was developed utilizing the cell-labeling dye CTY.
  • CTY cell-labeling dye
  • This dye enabled the quantification of T cell divisions, wherein every cell division resulted in a reduction in the amount of dye in the daughter cells of about 50% compared to the parent cell as measured by flow cytometry.
  • the frequency of the most proliferative cells correlates well with gold- standard measures of T cell proliferation such as the proliferation index (FIG. 5A) and the absolute cell count (FIG. 5B).
  • Example 3 Methods to improve T cell function in an immunosuppressive environment.
  • the overexpression of a barcoded library of genes in primary T cells was previously utilized to identify key regulators of T cell proliferation and other effector functions (Legut, M. et al. Nature 603, 728-735 (2022)).
  • the following example provides substantial enhancements to this screening approach to better mimic the complex cellular environment found in clinical settings. These improvements have led to higher data quality and a robust discovery of immunosuppressive resistance genes (FIG. 6).
  • the method comprises the co-culture of stimulated, library-transduced T cells with immunosuppressive factors. This includes the incorporation of autologous immunosuppressive cell types and culturing both CD4+ and CD8+ T cells in the same assay.
  • CD4+ and CD8+ T cells are crucial for the clinical efficacy of cell therapy products. Therefore, the immunosuppressive resistance genes identified in the context of CD4+ and CD8+ co-culture may hold more clinical relevance than those identified in CD4+ and CD8+ T cells when tested separately (Sommermeyer, D. el al. Leukemia 30, 492- 500 (2016)).
  • the supraphysiological conditions for T cell stimulation in previous approaches made it impossible to measure the effect of immunosuppression.
  • cells were previously cultured in the presence of 400 lU/mL of IL-2 to drive T cell proliferation in culture.
  • This level of IL-2 stimulation could have prevented the immunosuppressive activities of adenosine, TGF-P, regulatory T cells, or immunosuppressive macrophages described above. Consequently, conditions were established to both activate T cells in culture while enabling the controlled immunosuppressive effects of adenosine, TGF- P, regulatory T cells, and immunosuppressive macrophages in a physiological manner (FIGs. 7A, 7B).
  • Such conditions include the reduction in IL-2 supplementation during culture from a concentration of 400 lU/mL to a concentration of 50 lU/mL.
  • the methods described herein facilitated the execution of multiple screens per healthy T cell donor (FIGs. 8A, 8B). Coverage is a critical factor for robust data capture and maintaining reliable signal to noise ratios in pooled genomic screens (Sanjana, N. E. Anal. Biochem. 532, 95-99 (2017)). Furthermore, the capability to conduct multiple screens from the same donor enables the identification of immunosuppressive resistance genes that protect T cells from various forms of immunosuppression.
  • Example 2 The immunosuppression assays described in Example 2 were effectively scaled to enable genome-scale screens. A total of 12 screens (3 healthy donors, 4 immunosuppressive mechanisms) comprising co-culture of both CD4+ and CD8+ T cells were conducted. Across all screens, a robust suppression of T cell proliferation was observed (FIG. 9). The modifications to the previous platform described above led to a marked improvement in data quality, as measured by increased barcode recovery in the most proliferative T cell populations (FIG. 10).
  • Example 4 Methods and compositions for improving T cell tumor-killing capacity in an immunosuppressive environment.
  • immunosuppressive resistance genes identified by genome-scale screens in the immunosuppressive contexts were cloned from the screening library or synthesized. For expression in T cells, the immunosuppressive resistance genes were cloned into a lentiviral or retroviral vector which typically encoded a marker gene for selection of transduced cells.
  • Example selection markers include puromycin N-acetyltransferase, blasticidin-S deaminase, truncated nerve growth factor receptor, or truncated epidermal growth factor receptor.
  • the vector also encoded a CAR, for instance a CAR targeting mesothelin, HER2, or claudin 6 (CEDN6) as described, for example in WO 2015/090230 Al, WO 2017/079694 A2, or US 2022/0184119 Al, all of which are herein incorporated by reference in their entireties.
  • a CAR for instance a CAR targeting mesothelin, HER2, or claudin 6 (CEDN6) as described, for example in WO 2015/090230 Al, WO 2017/079694 A2, or US 2022/0184119 Al, all of which are herein incorporated by reference in their entireties.
  • the vector encoded a previously described gene in the context of CAR T cell armoring, such as cJUN, membrane bound IE15 or dominant negative TGF-P receptor 2 instead of immunosuppressive resistance genes.
  • CAR T cell armoring standards are described, for example, in WO 2023/172514 Al, US 2022/0307039 Al, and US 2023/0220343 Al, all of which are herein incorporated by reference in their entireties.
  • the transgenes were separated with self-cleaving 2A peptides such as P2A or T2A.
  • the expression was driven by the short EF-loc or SFFV promoters.
  • retroviral vectors the expression was driven by a MoMLV-based promoter.
  • the immunosuppressive resistance genes were delivered to activated primary human T cells (total CD3+, CD4+, CD8+ or gamma delta T cells).
  • the cells were either transduced with a tricistronic vector encoding the CAR, immunosuppressive resistance gene and a marker gene (see, e.g., FIGs. 15-19), or co-transduced with two bicistronic vectors, one encoding a CAR and a marker gene, and the other encoding a immunosuppressive resistance gene and another marker gene (see, e.g., FIGs. 11-14B).
  • the sequences of immunosuppressive resistance genes are listed in the Sequence Table below.
  • the transduced cells were characterized in terms of their cytotoxic activity against cancer cells expressing the CAR target.
  • the co-culture also included immunosuppressive factors such as polarized macrophages, regulatory T cells, adenosine or TGF-
  • a three-component co-culture system was applied to model the tumor microenvironment in vitro'.
  • Three-component co-cultures included CAR T cells expressing immunosuppressive resistance genes, tumor cells, and components of the immunosuppressive microenvironment (e.g., macrophages, Tregs, adenosine or TGF-
  • immunosuppressive resistance genes e.g., macrophages, Tregs, adenosine or TGF-
  • 3 regulatory T cell
  • CD4+ and CD8+ T cells co-expressing a HER-2-specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio.
  • Tested immunosuppressive resistance genes include FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL, the nucleotide and amino acid sequences of which are listed in the Sequence Table below. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). As shown in FIG.
  • immunosuppressive resistance genes COPZ2, GNL3, LIMA1, and LTBR the nucleotide and amino acid sequences of which are listed in the Sequence Table below, were next tested for their effects on CAR-T tumor-killing capacity in adenosine immunosuppression in vitro.
  • ectopic expression of these immunosuppressive resistance genes similarly resulted in increased tumor killing modified CAR-T cells in the presence of adenosine immunosuppression compared to unmodified CAR-T cell controls.
  • immunosuppressive resistance genes CD47, CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC STK11, YBX2, and ZTBTB46 were tested for their effects on CAR-T tumor-killing capacity in macrophage- mediated immunosuppression in vitro with the same tumor-killing assay.
  • ectopic expression of these immunosuppressive resistance genes also resulted in increased tumor killing capacity of modified CAR-T cells in the presence of macrophage-mediated immunosuppression compared to unmodified CAR-T cell controls.
  • T cell exhaustion poses another major limitation to the efficacy of solid tumor cell therapies.
  • a similar tumor-killing assay was performed wherein a T cells comprising a CAR targeting a solid tumor antigen were repeatedly challenged with cancer cells to recapitulate the process leading to T cell exhaustion in patients.
  • CD4+ and CD8+ T cells co-expressing a HER2-specific CAR and ZBTB46, the nucleotide and amino acid sequence of which is listed in the Sequence Table below, or a control irrelevant gene tNGFR were co-incubated with GFP-engineered HER2+ cancer cell lines at 1:4 T cells to cancer cells ratio.
  • Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to account for the presence of a specified immunosuppressive factor on cancer cell growth. As shown in FIG. 14A, ectopic expression of ZTBTB46 resulted in maintenance of stronger cytotoxic activity against cancer cells throughout the length of the cancer cell challenge in a normal culture background, while FIG. 14B similarly shows increased maintenance of tumor killing capacity of modified CAR-T cells in a TGF- P immunosuppressive culture environment.
  • LTBR coexpression with a HER2 CAR utilizing a CD28-based costimulatory domain enabled the T cells to clear multiple cancer challenges, to a much stronger extent than the other benchmark armoring genes.
  • CAR T cells co-expressing a control gene tEGFR (“Unmodified CAR”) or armoring gene LTBR were co-incubated with GFP+ SKOV3 ovarian cancer cells at 1:16 effector: target ratio for up to 140 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone. As shown in FIG. 23, armoring with LTBR enhanced tumor killing capacity of CLDN6 cx
  • LTBR-del is a non-functional, truncated version of LTBR lacking the intracellular signaling domain, resulting in an inability to transmit signaling even when the extracellular part binds a ligand.
  • LTBR-expressing CAR T cells were also able to withstand immunosuppression, in particular mediated by Tregs, in the repeated cancer challenge context, notably outperforming CAR-T cells expressing benchmark armoring genes cJUN, mbIL15, and TGFBR2dn at the second cancer cell challenge.
  • Claudin-6 (CLDN6)-targeting CAR-T cells utilizing a 4- IBB costimulatory domain were co-expressed with LTBR and similarly tested for tumor killing capacity against the CLDN6-high PAI and CLDN6-med OV90 ovarian cancer cell lines using a similar serial challenge assay as described above.
  • CD4+ and CD8+ T cells co-expressing a CLDN6-specific CAR and LTBR or a control irrelevant gene were co- incubated with GFP-engineered CLDN6+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells, as indicated.
  • LTBR expression resulted in a similar improvement of cytotoxic activity in the repeated cancer challenge assay, a proxy for resistance to exhaustion, in the context of a CLDN6 CAR utilizing a 4- IBB costimulatory domain in the context of ovarian cancer lines expressing different levels of the target antigen.
  • y8 T cells co-expressing a CLDN6-specific CAR and LTBR or a control irrelevant gene were co-incubated with GFP-engineered CLDN6+ cancer cell line OV90. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. After 96 h of co-incubation, T cells were harvested and stained in the presence of counting beads to determine their expansion and exhaustion. Exhausted cells are defined as LAG3+ TIM3+.
  • ectopic LTBR expression in lymphocytes can potentially increase the number of patients amendable to this therapeutic intervention, since T cell efficacy at a lower cancer-antigen threshold would mean more patients qualify for this treatment.
  • LTBR expression can potentially lead to more durable therapeutic responses, as most CAR-based therapies are only able to target high antigen-expression cancer cells, thereby leading to incomplete or inefficient tumor killing capacities.
  • OV90 ovarian cancer cells were obtained from American type culture collection (ATCC) and were cultured per ATCC recommendations until ready for use in mouse models. Endogenous Claudin 6 expression was verified by standard flow cytometry techniques using commercially available antibodies. After OV90 cells were expanded in vitro, 6-8 week old, female, NOD.Cg-
  • T cell injection Following T cell injection, survival of the animals was monitored daily. Animals were removed from the study and euthanized upon reaching a tumor burden of over 2,000 mm 3 or another humane endpoint. * p ⁇ 0.05 (log-rank test). After reaching the experimental endpoint, explanted tumors were weighed and dissociated by enzymatic digestion. Equal numbers of cells were taken from each digested sample and were processed for flow cytometry analysis using standard techniques. To quantify cells in each sample, a fixed number of quantification beads were added. Tumor cells were identified as a human HLA-ABC positive and CD4/CD8 negative population. T cells were identified as human HLA-ABC and CD4/CD8 positive populations.
  • CAR-T cells co-expressing LTBR showed enhanced survival relative to both no CAR T cell control and CAR-T cells co-expressing the tEGFR control gene. No significant changes in mouse body weight were observed in the CAR-T + LTBR group relative to either control group (FIG. 20B).
  • matched tumor samples were explanted and processed to assess tumor infiltration by human T cells.
  • OV90 tumors in mice treated with CAR-T cells co-expressing LTBR exhibited increased tumor infiltration by human T cells relative to CAR_T cells co-expressing the control tEGFR gene.
  • FIG. 20D all in vivo tumor cells exhibited decreased CLDN6 levels relative to OV90 in vitro control cells, demonstrating that CAR-T cells co-expressing LTBR have increased ability to target antigen-low cancer cells in vivo relative to controls.
  • Embodiment 1 A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • Embodiment 2 The modified lymphocyte of Embodiment 1, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 3 The modified lymphocyte of Embodiment 1 or Embodiment 2, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 4 The modified lymphocyte of any one of Embodiments 1-3, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
  • Embodiment 5 The modified lymphocyte of any one of Embodiments 1-3, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, ADARB1, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63,
  • Embodiment 6 A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, C0PZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, C0PZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or
  • Embodiment 7 The modified lymphocyte of Embodiment 6, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 8 The modified lymphocyte of Embodiment 6 or Embodiment 7, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 9 A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the modified lymphocyte has increased proliferation and/or increased effector function in in vivo or in vitro tumor killing assays relative to an unmodified lymphocyte.
  • the immunosuppressive resistance gene is LTBR
  • the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof
  • the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein
  • the modified lymphocyte has increased proliferation and/or increased effector function in in vivo or in vitro tumor killing assays relative to an unmodified lymphocyte.
  • Embodiment 10 The modified lymphocyte of Embodiment 9, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding LTBR.
  • Embodiment 11 The modified lymphocyte of Embodiment 9 or Embodiment 10, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding LTBR.
  • Embodiment 13 The modified lymphocyte of any one of Embodiments 6-8, wherein the increased level of the immunosuppressive resistance gene results in increased tumor cell killing of the modified lymphocyte in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene.
  • Embodiment 14 The modified lymphocyte of any one of Embodiments 1-13, wherein the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6.
  • Embodiment 15 The modified lymphocyte of any one of Embodiments 1-14, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor
  • Embodiment 16 The modified lymphocyte of Embodiment 15, wherein the CAR or the TCR binds to a tumor antigen.
  • Embodiment 17 The modified lymphocyte of Embodiment 15 or Embodiment 16, wherein the increased level of the immunosuppressive resistance gene LTBR results in increased killing of cells expressing a low level of antigen in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene, wherein the antigen is bound by the CAR or the TCR.
  • Embodiment 18 The modified lymphocyte of Embodiment 17, wherein the tumor killing assay is an in vitro tumor killing assay.
  • Embodiment 19 The modified lymphocyte of any one of Embodiments 15-18, wherein the CAR or the TCR binds to HER2 or Claudin-6 (CLDN6).
  • Embodiment 20 The modified lymphocyte of any one of Embodiments 15-19, wherein the modified lymphocyte further comprises an expression cassette comprising a nucleic acid encoding the CAR or the TCR.
  • Embodiment 21 The modified lymphocyte of Embodiment 20, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in the same expression cassette.
  • Embodiment 22 The modified lymphocyte of Embodiment 20, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in separate expression cassettes.
  • Embodiment 23 The modified lymphocyte of any one of Embodiments 1-22, wherein exhaustion of the modified lymphocyte is reduced compared to a lymphocyte that does not express the immunosuppressive resistance gene.
  • Embodiment 24 The modified lymphocyte of any one of Embodiments 1-23, wherein the modified lymphocyte maintains the ability to kill tumor cells expressing a tumor antigen following at least two exposures to the tumor cells.
  • Embodiment 25 The modified lymphocyte of any one of Embodiments 1-24, wherein the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
  • Embodiment 26 The modified lymphocyte of any one of Embodiments 1-25, wherein the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs).
  • Embodiment 27 The modified lymphocyte of any one of Embodiments 1-26, wherein the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
  • TIL tumor infiltrating lymphocyte
  • Embodiment 28 The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
  • Embodiment 29 The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
  • Embodiment 30 The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a TCRy8+ lymphocyte.
  • Embodiment 31 The modified lymphocyte of Embodiment 30, wherein the modified lymphocyte is a TCRy8+ lymphocyte and is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR.
  • Embodiment 32 The modified lymphocyte of Embodiment 31, wherein the increased level of the immunosuppressive resistance gene LTBR results in increased killing of antigen-low cancer cells in tumor killing assays compared to an unmodified lymphocyte.
  • Embodiment 33 The modified lymphocyte of Embodiment 32, wherein the tumor killing assay is an in vitro tumor killing assay.
  • Embodiment 34 The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
  • TSCM stem cell-like memory
  • TCM central memory
  • TEM effector memory
  • TEMRA+ effector memory RA+
  • Embodiment 35 The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a regulatory T cell.
  • Embodiment 36 The modified lymphocyte of any one of Embodiments 1-35, wherein the modified lymphocyte is a human lymphocyte.
  • Embodiment 37 The modified lymphocyte of any one of Embodiments 1-36, wherein the modified lymphocyte is an autologous lymphocyte.
  • Embodiment 38 The modified lymphocyte of any one of Embodiments 3-37, wherein the modified lymphocyte comprises a vector comprising the expression cassette.
  • Embodiment 39 The modified lymphocyte of any one of Embodiments 3-38, wherein the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
  • Embodiment 40 The modified lymphocyte of Embodiment 39, wherein the promoter is a ubiquitous promoter.
  • the modified lymphocyte of Embodiment 40, wherein the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate- early enhancer and chicken beta-actin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
  • CMV cytomegalovirus
  • CAG chicken beta-actin
  • EFla elongation factor la
  • UbC ubiquitin C
  • 5’ LTR 5’ LTR
  • CMV CMV
  • Embodiment 42 The modified lymphocyte of Embodiment 39, wherein the promoter is an inducible promoter.
  • Embodiment 43 The modified lymphocyte of any one of Embodiments 39-42, wherein the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
  • Embodiment 44 The modified lymphocyte of any one of Embodiments 39-43, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte.
  • Embodiment 45 The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene.
  • Embodiment 46 The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus.
  • Embodiment 47 The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
  • Embodiment 48 A vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • Embodiment 49 The vector of Embodiment 48, wherein the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 50 The vector of Embodiment 48 or Embodiment 49, further comprising nucleic acid encoding a therapeutic protein.
  • Embodiment 51 The vector of Embodiment 50, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • Embodiment 52 The vector of Embodiment 51, wherein the nucleic acid encoding the
  • CAR or TCR are included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 53 The vector of Embodiment 51, wherein the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or TCR are included in a second expression cassette.
  • Embodiment 54 The vector of any one of Embodiments 48-53, wherein the vector is a viral vector.
  • Embodiment 55 The vector of any one of Embodiments 48-54, wherein the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
  • Embodiment 56 The vector of Embodiment 55, the vector is an episomal or nonintegrating vector.
  • Embodiment 57 The vector of Embodiment 56, wherein the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
  • SV40 Simian virus 40
  • Adenovirus Adenovirus
  • Adeno-associated vector Adeno-associated vector
  • Embodiment 58 The vector of any one of Embodiments 48-53, wherein the vector is a non-viral vector.
  • Embodiment 59 The vector of Embodiment 58, wherein the non-viral vector is a plasmid.
  • Embodiment 60 The vector of any one of Embodiments 48-59, wherein the vector further comprises nucleic acid encoding a drug-resistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
  • Embodiment 61 A modified lymphocyte comprising one or more of the vectors as defined in any one of Embodiments 48-60.
  • Embodiment 62 A composition comprising the modified lymphocyte as defined in any one of Embodiments 1-47.
  • Embodiment 63 The composition of Embodiment 62, wherein the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
  • Embodiment 64 A method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the vector of any one of Embodiments 48-60.
  • Embodiment 65 The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a tumor microenvironment.
  • Embodiment 66 The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
  • Embodiment 67 The method of Embodiment 66, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, ADARB1, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, A
  • Embodiment 68 The method of Embodiment 66 or Embodiment 67, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR.
  • Embodiment 69 The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment.
  • Embodiment 70 The method of Embodiment 69, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, AL0X15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, AP0A5, AP0C4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPI
  • Embodiment 71 The method of Embodiment 69 or Embodiment 70, wherein the immunosuppressive resistance gene is ZBTB46.
  • Embodiment 72 The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
  • Embodiment 73 The method of Embodiment 72, wherein the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD 10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, AD ATI, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP
  • Embodiment 74 The method of Embodiment 72 or Embodiment 73, wherein the immunosuppressive resistance gene is selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL.
  • Embodiment 75 The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment.
  • Embodiment 76 The method of Embodiment 75, wherein the immunosuppressive resistance gene is selected from the group consisting of AB AT, AB HD 12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, AL0XE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARE4D, ARMC2, ARNTE, ASAP3, ASB3, ASIC2, ASPH, ASTE1,
  • Embodiment 78 A method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of:
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i),
  • Embodiment 79 The method of Embodiment 78, wherein the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Embodiment 80 The method of Embodiment 78 or Embodiment 79, wherein the exogenous nucleic acid further comprises a T cell receptor (TCR).
  • TCR T cell receptor
  • Embodiment 81 The method of any one of Embodiments 78-80, wherein the peripheral blood mononuclear cells are obtained from leukapheresis.
  • Embodiment 82 The method of any one of Embodiments 78-81, wherein the CD8+ and CD4+ T cells are isolated sequentially.
  • Embodiment 83 The method of any one of Embodiments 78-82, wherein the naive CD4+ T cells are differentiated into activated CD4+ T cells.
  • Embodiment 84 The method of any one of Embodiments 78-83, wherein the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
  • Embodiment 85 The method of any one of Embodiments 78-84, wherein the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
  • Embodiment 86 The method of any one of Embodiments 78-85, wherein the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
  • Embodiment 87 The method of any one of Embodiments 78-86, wherein the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
  • Embodiment 88 The method of any one of Embodiments 78-87, wherein the transduced lymphocytes are enriched by positive selection.
  • Embodiment 89 The method of Embodiment 88, wherein the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
  • Embodiment 90 A method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising:
  • lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual
  • Embodiment 91 The method of Embodiment 90, wherein the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
  • Embodiment la A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
  • Embodiment 2a The modified lymphocyte of Embodiment la, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 3a The modified lymphocyte of Embodiment la or Embodiment 2a, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 4a The modified lymphocyte of any one of Embodiments la-3a, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
  • Embodiment 5a The modified lymphocyte of any one of Embodiments la-3a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, ADARB1, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP
  • Embodiment 6a The modified lymphocyte of any one of Embodiments la-5a, wherein increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a tumour microenvironment compared to an unmodified lymphocyte.
  • Embodiment 7a The modified lymphocyte of any one of Embodiments la-6a, wherein the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6.
  • Embodiment 8a The modified lymphocyte of any one of Embodiments la-7a, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • Embodiment 9a The modified lymphocyte of Embodiment 8a, wherein the lymphocyte further comprises an expression cassette comprising the nucleic acid encoding the CAR or TCR.
  • Embodiment 10a The modified lymphocyte of Embodiment 8a or Embodiment 9a, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in the same expression cassette.
  • Embodiment I la The modified lymphocyte of Embodiment 8a or Embodiment 9a, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in separate expression cassettes.
  • Embodiment 12a The modified lymphocyte of any one of Embodiments la-1 la, wherein the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
  • Embodiment 13a The modified lymphocyte of any one of Embodiments la- 12a, wherein the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • Embodiment 14a The modified lymphocyte of any one of Embodiments la- 13a, wherein the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
  • TIL tumor infiltrating lymphocyte
  • Embodiment 15a The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
  • Embodiment 16a The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
  • Embodiment 17a The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a TCRy8+ lymphocyte.
  • Embodiment 18a The modified lymphocyte of any one of Embodiments la-14a, wherein the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
  • TSCM stem cell-like memory
  • TCM central memory
  • TEM effector memory
  • TEMRA+ effector memory
  • Embodiment 19a The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a regulatory T cell.
  • Embodiment 20a The modified lymphocyte of any one of Embodiments la- 19a, wherein the modified lymphocyte is a human lymphocyte.
  • Embodiment 21a The modified lymphocyte of any one of Embodiments la- 20a, wherein the modified lymphocyte is an autologous lymphocyte.
  • Embodiment 22a The modified lymphocyte of any one of Embodiments 3a-21a, wherein the modified lymphocyte comprises a vector comprising the expression cassette.
  • Embodiment 23a The modified lymphocyte of any one of Embodiments 3a- 22a, wherein the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
  • Embodiment 24a The modified lymphocyte of Embodiment 23a, wherein the promoter is a ubiquitous promoter.
  • Embodiment 25a The modified lymphocyte of Embodiment 24a, wherein the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate- early enhancer and chicken beta-actin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
  • CMV cytomegalovirus
  • CAG chicken beta-actin
  • EFla elongation factor la
  • UbC ubiquitin C
  • 5’ LTR 5’ LTR
  • CMV cytomegalovirus
  • Embodiment 26a The modified lymphocyte of Embodiment 23a, wherein the promoter is an inducible promoter.
  • Embodiment 27a The modified lymphocyte of any one of Embodiments 23a-26a, wherein the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
  • Embodiment 28a The modified lymphocyte of any one of Embodiments 23a- 27a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte.
  • Embodiment 29a The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene.
  • Embodiment 30a The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus.
  • Embodiment 31a The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
  • Embodiment 32a A vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
  • Embodiment 33a The vector of Embodiment 32a, wherein the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 34a The vector of Embodiment 32a or Embodiment 33a, further comprising nucleic acid encoding a therapeutic protein.
  • Embodiment 35a The vector of Embodiment 34a, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • Embodiment 36a The vector of Embodiment 35a, wherein the nucleic acid encoding the CAR or TCR are included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
  • Embodiment 37a The vector of Embodiment 35a, wherein the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or TCR are included in a second expression cassette.
  • Embodiment 38a The vector of any one of Embodiments 32a-37a, wherein the vector is a viral vector.
  • Embodiment 39a The vector of any one of Embodiments 32a-38a, wherein the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
  • Embodiment 40a The vector of Embodiment 39a, the vector is an episomal or nonintegrating vector.
  • Embodiment 41a The vector of Embodiment 40a, wherein the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
  • SV40 Simian virus 40
  • Adenovirus Adenovirus
  • Adeno-associated vector Adeno-associated vector
  • Embodiment 42a The vector of any one of Embodiments 32a-37a, wherein the vector is a non-viral vector.
  • Embodiment 43a The vector of Embodiment 42a, wherein the non-viral vector is a plasmid.
  • Embodiment 44a The vector of any one of Embodiments 32a-43a, wherein the vector further comprises nucleic acid encoding a drug-resistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
  • Embodiment 45a A modified lymphocyte comprising one or more of the vectors as defined in any one of Embodiments 32a-44a.
  • Embodiment 46a A composition comprising the modified lymphocyte as defined in any one of Embodiments la-3 la.
  • Embodiment 47a The composition of Embodiment 46a, wherein the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
  • Embodiment 48a A method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the vector of any one of Embodiments 32a-44a.
  • Embodiment 49a The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a tumor microenvironment.
  • Embodiment 50a The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
  • Embodiment 51a The method of Embodiment 50a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, ADARB1, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, A
  • Embodiment 53a The method of Embodiment 52a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA
  • Embodiment 54a The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
  • Embodiment 55a The method of Embodiment 54a, wherein the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD 10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, AD ATI, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2,
  • Embodiment 57a The method of Embodiment 56a, wherein the immunosuppressive resistance gene is selected from the group consisting of AB AT, AB HD 12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, ALOXE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A
  • Embodiment 58a A method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of:
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i),
  • Embodiment 59a The method of Embodiment 58a, wherein the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Embodiment 60a The method of Embodiment 58a or Embodiment 59a, wherein the exogenous nucleic acid further comprises a T cell receptor (TCR).
  • TCR T cell receptor
  • Embodiment 61a The method of any one of Embodiments 58a-60a, wherein the peripheral blood mononuclear cells are obtained from leukapheresis.
  • Embodiment 62a The method of any one of Embodiments 58a-61a, wherein the CD8+ and CD4+ T cells are isolated sequentially.
  • Embodiment 63a The method of any one of Embodiments 58a-62a, wherein the naive CD4+ T cells are differentiated into activated CD4+ T cells.
  • Embodiment 64a The method of any one of Embodiments 58a-63a, wherein the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
  • Embodiment 65a The method of any one of Embodiments 58a-64a, wherein the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
  • Embodiment 66a The method of any one of Embodiments 58a-65a, wherein the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
  • Embodiment 67a The method of any one of Embodiments 58a-66a, wherein the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
  • Embodiment 68a The method of any one of Embodiments 58a-67a, wherein the transduced lymphocytes are enriched by positive selection.
  • Embodiment 69a The method of Embodiment 68a, wherein the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
  • Embodiment 70a A method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising:
  • lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual
  • Embodiment 71a The method of Embodiment 70a, wherein the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present disclosure relates in some aspects to modified lymphocytes capable of resisting immunosuppressive cellular environments.

Description

METHODS AND COMPOSITIONS FOR IMPROVING T CELL FUNCTION IN AN IMMUNOSUPPRESSIVE ENVIRONMENT
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/558,573, filed on February 27, 2024, entitled “METHODS AND COMPOSITIONS FOR IMPROVING T CELL FUNCTION IN AN IMMUNOSUPPRESSIVE ENVIRONMENT”, which is herein incorporated by reference in its entirety for all purposes.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The content of the electronic sequence listing (304952000140seqlist.xml; Size: 86,112 bytes; and Date of Creation: February 13, 2025) is herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present disclosure relates in some aspects to modified lymphocytes capable of resisting immunosuppressive cellular environments.
BACKGROUND
[0004] Cellular immunotherapies with engineered autologous patient T-cells redirected against a chosen tumor antigen have yielded great efficacy against blood cancers, resulting in six FDA approvals for chimeric antigen receptors (CARs) to date. In contrast, CAR therapy for solid tumors has shown overall much lower efficacy, due to suppression of T-cell effector function in the tumor microenvironment (Singh, Nathan, and Marcela V Maus. Immunity vol. 56,10 (2023): 2296-2310). The immunosuppressive environment is also a significant contributor to blood cancer relapse after treatment with FDA-approved CAR therapies. For instance, the frequency of regulatory T cells (Tregs) predicts relapse of lymphoma patients treated with axi-cel and tisa-cel (Haradhvala, Nicholas J et al. Nature medicine vol. 28,9 (2022): 1848-1859 and Good, Zinaida et al. Nature medicine vol. 28,9 (2022): 1860-1871). Similarly, the initial efficacy of tumor-infiltrating lymphocytes (TIL) in solid tumors can be boosted by total body irradiation which temporarily depletes Tregs. Conversely, an influx of Tregs and tumor-infiltrating macrophages was shown to suppress CAR T cell activity in multiple solid tumors (O'Rourke, Donald M et al. Science translational medicine vol. 9,399 (2017): eaaa0984). Thus, in some aspects, existing cellular therapies to treat cancer are limited due to the immunosuppressive environment present within solid tumors. Improved T cell therapies that can resist immunosuppression are therefore needed. Provided herein are methods and compositions that address such and other needs.
SUMMARY
[0005] In some aspects, provided herein is a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
[0006] In some embodiments, the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
[0007] In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
[0008] In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, G0RASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
[0009] In some aspects, provided herein is a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of MCAM, FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
[0010] In some aspects, provided herein is a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, HOXB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, ORM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PL0D2, PLPPR2, PNPT1, P0LR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYR0XD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL3O, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN1O, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
[0011] In some aspects, provided herein is a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, COPZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
[0012] In some embodiments, the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
[0013] In some aspects, provided herein is a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein. [0014] In some embodiments, the modified lymphocyte comprises an exogenous nucleic acid encoding LTBR. In some embodiments, the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding LTBR.
[0015] In some embodiments, the increased level of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a tumour microenvironment compared to an unmodified lymphocyte.
[0016] In some embodiments, the increased level of the immunosuppressive resistance gene results in increased tumor cell killing of the modified lymphocyte in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene. [0017] In some embodiments, the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6.
[0018] In some embodiments, the nucleic acid further encodes at least two immunosuppressive resistance genes selected from the group consisting of FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA. [0019] In some embodiments, the modified lymphocyte expresses the therapeutic protein.
[0020] In some embodiments, the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR or the TCR binds to a tumor antigen.
[0021] In some embodiments, the increased level of LTBR results in increased killing of cells expressing a low level of antigen in a tumor killing assay compared to a lymphocyte that does not express the LTBR, wherein the therapeutic protein is a CAR or a TCR, wherein the antigen is bound by the CAR or the TCR. In some embodiments, the tumor killing assay is an in vitro tumor killing assay.
[0022] In some embodiments, the CAR or the TCR binds to HER2 or Claudin-6 (CLDN6). [0023] In some embodiments, the modified lymphocyte further comprises an expression cassette comprising the nucleic acid encoding the CAR or the TCR. In some embodiments, the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in the same expression cassette. In some embodiments, the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in separate expression cassettes. [0024] In some embodiments, exhaustion of the modified lymphocyte is reduced compared to a lymphocyte that does not express the immunosuppressive resistance gene.
[0025] In some embodiments, the modified lymphocyte maintains the ability to kill tumor cells expressing a tumor antigen following at least two exposures to the tumor cells.
[0026] In some embodiments, the modified lymphocyte is a T cell, a NK cell, or a NK T cell. In some embodiments, the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs). In some embodiments, the modified lymphocyte is a tumor infiltrating lymphocyte (TIL). In some embodiments, the modified lymphocyte is a CD4+ T cell or a CD8+ T cell. In some embodiments, the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
[0027] In some embodiments, the modified lymphocyte is a TCRy5+ lymphocyte. In some embodiments, the modified lymphocyte is a TCRy5+ lymphocyte and is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR. In some embodiments, the increased level of the immunosuppressive resistance gene LTBR results in increased killing of antigen-low cancer cells in tumor killing assays compared to an unmodified lymphocyte. In some embodiments, the tumor killing assay is an in vitro tumor killing assay.
[0028] In some embodiments, the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell. In some embodiments, the modified lymphocyte is a regulatory T cell. In some embodiments, the modified lymphocyte is a human lymphocyte. In some embodiments, the modified lymphocyte is an autologous lymphocyte. In some embodiments, the modified lymphocyte comprises a vector comprising the expression cassette.
[0029] In some embodiments, the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate-early enhancer and chicken betaactin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte. [0030] In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus. In some embodiments, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
[0031] In other aspects, provided herein is a vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
[0032] In other aspects, provided herein is a vector comprising nucleic acid encoding an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, and wherein the immunosuppressive resistance gene is selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, VEGFA, ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS1L, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, G0RASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
[0033] In some embodiments, the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the vector further comprises nucleic acid encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the nucleic acid encoding the CAR or the TCR is included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or the TCR is included in a second expression cassette. [0034] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus. In some embodiments, vector is an episomal or non-integrating vector. In some embodiments, the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
[0035] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid.
[0036] In some embodiments, the vector further comprises nucleic acid encoding a drugresistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
[0037] In other aspects, provided herein is a modified lymphocyte comprising one or more of the vectors described herein.
[0038] In other aspects, provided herein is a composition comprising the modified lymphocyte as defined herein. In some embodiments, the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
[0039] In other aspects, provided herein is a method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the any vector described herein.
[0040] In other aspects, provided herein is a method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising introducing into lymphocytes the vector of any one of claims 44-56 or increasing expression of an immunosuppressive resistance gene selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, VEGFA, ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CA13, CALC0C01, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS1L, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, HOXB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
[0041] In some embodiments, the immunosuppressive cellular environment comprises a tumor microenvironment.
[0042] In some embodiments, the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of COPZ2, ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, AD ARBI, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP6V1C1, ATP6V1D, ATPAF1, ATRAID, ATRIP, AUTS2, AZI2, B3GNT9, BAIAP2, BAIAP2L2, BAP1, BATE, BCAT1, BCKDHA, BCL2L13, BCL2L2, BLMH, BMPER, BOD1, BPIFA1, BSG, BSPRY, BTC, BTD, BTRC, C18orf54, C1QTNF12, C1QTNF4, C3orf38, C6orf223, C8orf76, C9orfll6, CALCOCO1, CALHM1, CAT, CAVIN1, CAVIN4, CBWD1, CCDC113, CCDC121, CCDC14, CCDC174, CCDC86, CCDC93, CCIN, CCN2, CCN3, CCR6, CCSAP, CCT7, CD19, CD1B, CD36, CD46, CD86, CD96, CDC14A, CDH1, CDK2AP1, CDK7, CELA2B, CEP97, CFAP46, CFAP47, CHAMP1, CHMP2B, CHST8, CKB, CLEC11A, CLEC4M, CLEC9A, CLPB, CLPSL1, CLSTN1, CLUL1, CLYBL, CNDP2, CNTROB, COA6, COL15A1, COL21A1, COL22A1, COLQ, COMMD5, CORO6, COX11, CPA1, CPSF6, CPT2, CRB3, CREB3L2, CREM, CRHR2, CRTC2, CRYGD, CSK, CTBP2, CTNNA2, CTSS, CX3CR1, CYP20A1, CYP2C9, DADI, DAXX, DBF4, DCDC2, DCLK2, DCTN3, DCX, DDB1, DDHD1, DDO, DDOST, DDX21, DDX56, DEGS2, DEPDC7, DFFB, DIXDC1, DLK1, DNAI2, DNAJC16, DPPA4, DPYSL3, DPYSL4, DRG2, DUT, E2F7, EBNA1BP2, ECHDC1, ECHDC3, ECU, ECRG4, EDC3, EFCAB1, EFCAB12, EFCAB13, EFEMP2, EGR2, EIF2A, EIF4G3, EMP1, ENPP3, EPHA4, EPHB6, EPX, ERCC2, ERN2, ERV3-1, ESRP1, ETNK2, EVI2B, EXO1, EXTL3, EZH2, F10, F13B, F2, FAF2, FAM110A, FAM13C, FAM161B, FAM181A, FAM200A, FAM71A, FAM98A, FANCB, FATE1, FBXL13, FBXL16, FBXL20, FCRLB, FEM1C, FEV, FGF22, FGF3, FKBP14, FKBP5, FKBPL, FLOT1, FLVCR1, FLYWCH1, FMN1, FMO5, FOSB, FOXJ1, FOXS1, FPGT, FRMD5, FUT8, G6PC3, GAB3, GABRA5, GABRB2, GABRQ, GAD2, GALNT7, GAN, GAPDHS, GAPVD1, GBP4, GCNA, GDF6, GDF9, GDPD5, GET1, GET4, GGA1, GGA2, GGTLC1, GIMAP8, GKAP1, GLRB, GLT6D1, GLTPD2, GNAI2, GNL3, G0RASP1, GORASP2, GOSR1, GPAA1, GPC5, GPR119, GPR37, GPR65, GPRASP2, GPRC5C, GPT2, GPX1, GRHL2, GRID1, GRINA, GSDME, GTF2H1, GTF2I, GTPBP2, GUSB, GYPA, H2BC4, HABP4, HACD3, HAGH, HAND2, HARS2, HAUS7, HAX1, HDAC9, HDHD3, HENMT1, HEPACAM2, HHEX, HIBCH, HID1, HK1, HLA-C, HLCS, HNF4A, HNRNPAB, HNRNPD, H0XA11, H0XD8, HP1BP3, HPS3, HPS4, HSDL1, HSP90B1, HTR1F, HTRA4, HVCN1, IBSP, IDS, IFNA8, IFNAR2, IFNGR1, IFNLR1, IGFBP1, IGKV1D-17, IHO1, IK, IL10RA, IL12RB2, IL13RA2, IL17RE, IL36G, IMPDH2, INF2, INKAI, INTS5, IP6K2, IPO5, IQCA1, IREB2, JAGN1, JMJD8, KAT14, KAT7, KAZALD1, KCNAB3, KCNG3, KCNJ14, KCNK12, KCNK4, KCNK9, KCNMB2, KCNV1, KCTD4, KHDRBS2, KHDRBS3, KIAA0930, KIF7, KIFC3, KLHL14, KLHL2, KLHL8, KREMEN2, KRT13, KRT79, LARS2, LCK, LECT2, LGALS12, LHX4, LIMA1, LMX1B, LNX1, LOXL1, LRP3, LRP5L, LRRC18, LRRC45, LRRC55, LRRC61, LRRN3, LTBR, LYVE1, MAFB, MAG, MAGT1, MANSC1, MAP1LC3C, MAP2K1, MAP2K4, MAP2K5, MAP6D1, MAPK14, MARVELD2, MCAM, MCF2L, MCM10, MCOLN3, MED1, MEM01, METTL14, MEX3B, MFAP4, MGAT4B, MGME1, MICU2, MIER3, MKS1, MLLT11, MLST8, MMP13, MOBP, MOVIO, MRPL3, MRPS27, MSTN, MTG2, MTHFD2, MTMR12, MTMR8, MYBL1, MYCL, MYO1A, MYORG, NACC2, NADSYN1, NAE1, NBPF15, NCAM1, NCDN, NCLN, NDUFA2, NDUFB7, NECAP1, NECAP2, NECTIN1, NECTIN2, NEK3, NF2, NFIL3, NGFR, NIBAN1, NID2, NIF3L1, NIPSNAP3B, NKX2-8, NOA1, NOXI, NPLOC4, NPNT, NR1I2, NRM, NRTN, NT5DC1, NTPCR, NUP37, NXF3, NXPE3, NXT1, OAT, OIT3, OLFM2, OR1F1, OR1N1, OR52E8, OR9A4, 0RM2, OTUD7B, OTX1, OXCT2, P2RX4, P2RX5, P2RX7, P2RY6, P2RY8, P4HTM, PABPC3, PAF1, PAIP2, PARD6B, PARVA, PAX9, PCCA, PCDHGB1, PCLO, PDE1A, PDE4A, PDE9A, PDGFRA, PDHB, PDSS1, PDZD3, PDZD9, PELO, PFKL, PFKP, PI3, PI4K2A, PIGO, PIM1, PIMREG, PLA2G7, PLAGL1, PLAT, PLEKHO2, PLOD1, PLOD2, PLPP1, POFUT2, POGLUT2, POLR1C, POLR2L, POLR3C, POU3F2, PPA2, PPHLN1, PPP1R16B, PPP1R32, PPP1R9B, PPP2R2B, PPP2R3B, PRAME, PRDX3, PREB, PRELP, PRKCE, PRKCH, PRMT8, PRSS3, PSMB10, PSMC4, PSMD3, PTK6, PTPN18, PTPN2, PTPN9, PYROXD2, R3HDM2, RAB29, RAB30, RAB33B, RAB6B, RAB8B, RAD18, RAET1E, RAG2, RASGRP4, RASSF1, RBM12, RBM46, RCBTB2, RCC1L, RDX, RECQL4, REEP4, REG3A, RET, RGCC, RILP, RIMS2, RIN1, RINL, RITA1, RMND5A, RNF181, RPA3, RPL18, RPL28, RPS6KA2, RRM2, RRP15, RTN2, RUFY4, RUNDC1, SAMD13, SAMD4A, SCEL, SCGB2A2, SDCBP, SDHAF2, SEC23B, SEC63, SEMA3D, SEMA6D, SENP5, SEPTIN6, SEPTIN7, SERPINA10, SERPINE2, SFT2D2, SGCA, SGCB, SGIP1, SH3GL2, SHFL, SHOC2, SIAH2, SIGLEC7, SIRPG, SIRT6, SLC17A4, SLC22A5, SLC23A1, SLC23A3, SLC25A25, SLC25A3, SLC25A40, SLC25A41, SLC27A2, SLC27A3, SLC29A4, SLC2A8, SLC37A2, SLC39A12, SLC41A3, SLC44A5, SLC46A3, SLC51B, SLC6A5, SLCO4A1, SMAD3, SMG9, SMIM19, SMOX, SMPDL3B, SNF8, SNX5, SOCS6, SOD3, SOHLH2, SOX6, SOX8, SOX9, SPAG5, SPATA4, SPATCI, SPNS1, SPOCK1, SPRED1, SPSB1, SRF, SRM, SRPK2, SRRD, SSH3, SSRP1, ST6GALNAC6, ST7, ST8SIA3, STAT4, STIMATE, STING1, STOML2, STX1B, STX5, STXBP2, SYCE1, SYT11, SYT9, TAB2, TAMM41, TBC1D21, TBCD, TBL3, TBRG4, TBX10, TBX21, TBX22, TCF7L2, TERF2IP, TES, TEX10, TEX2, TEX45, TFAP2A, TFF2, TGFB3, THOC1, THOC3, THOP1, THRB, THRSP, THUMPD3, TIPRL, TLE1, TLE4, TMED3, TMEM107, TMEM183A, TMEM64, TMEM98, TMLHE, TMOD4, TNFSF15, TNNT2, TOMM22, TPRG1L, TPST2, TRAF3IP1, TRIM40, TRIM47, TRIP13, TRMT13, TRMT1L, TRMT2A, TRPV2, TRPV5, TSGA10, TSPAN16, TSPAN2, TSPAN32, TSPAN4, TSPAN5, TUBAL3, TUBB, TUBB3, TUBB4A, TUBGCP2, TULP4, TUT7, TXNIP, U2AF1L4, UBA6, UBASH3B, UBE2B, UBE2G1, UBE2I, UBE2O, UBQLN2, UBR2, UGDH, ULK3, UMOD, UNC13D, UQCRC2, UQCRFS1, USP1O, USP28, USP33, UVRAG, VAX2, VEGFA, VNN1, VNN3, VPS72, VRTN, VSIG8, VTI1B, VWC2, WBP11, WDFY2, WDR4, WDR60, WDTC1, WEE1, WFDC13, WFDC6, WNT2B, WRAP73, WSB1, XKR6, XPO7, XRCC5, XXYLT1, YAP1, YBX3, YY1AP1, ZBTB18, ZBTB39, ZDHHC20, ZDHHC5, ZIK1, ZKSCAN1, ZNF101, ZNF16, ZNF175, ZNF224, ZNF25, ZNF334, ZNF350, ZNF462, ZNF485, ZNF519, ZNF653, ZNF677, ZNF71, ZNF761, ZNF776, ZNF785, ZNHIT2, and ZSCAN18. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR.
[0043] In some embodiments, the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, Clorf35, Clorf87, C1QB, C1QL3, C1QTNF12, C3AR1, C7orf26, CACNG3, CACNG6, CAFHM1, CAFHM3, CAPN2, CAPZA2, CARD8, CASD1, CASK, CATSPER2, CAVIN3, CCDC106, CCDC121, CCDC153, CCDC68, CCDC70, CCDC93, CCE28, CCNE1, CCNY, CCT7, CD300C, CD300ED, CD96, CDC37, CDC42EP2, CDCA2, CDH13, CDH26, CDHR1, CDPF1, CDRT4, CEP120, CEP43, CERKE, CERS2, CES2, CFAP161, CFAP46, CGGBP1, CHID1, CHKA, CHRND, CIART, CITED2, CITED4, CLDN18, CLEC2D, CLSTN1, CLSTN3, CNDP2, COL6A2, C0LCA2, COMT, COPZ2, CORO6, CPA6, CPT1C, CREB3L2, CRLF3, CRP, CSH1, CYP2C8, CYP2D6, CYP51A1, DBT, DCDC2, DCP2, DCUN1D5, DDB1, DDI2, DDX1, DDX53, DEAF1, DELEI, DEXI, DGKG, DHRS13, DKK4, DNAJB8, DNAJC11, DNTT, DOK5, DPP4, DPYSL4, DPYSL5, DSCC1, DUSP8, DYDC2, E2F2, EBP, ECU, EDN3, EGLN3, EIF1AY, EIF2AK1, ELAVL1, ELN, ELOA, ELOA2, EMC3, EMC4, ENKUR, ENOXI, EPB41L1, EPB41L4A, EPHX4, ERICH1, ESRP1, ETFDH, FAM161B, FAM207A, FAM20C, FAM76B, FAM78A, FBXL3, FBXO31, FBXO4, FCGR2A, FCN2, FCRL5, FCRLB, FGF18, FGFR1OP2, FGL1, FHL5, FIGNL1, FLU, FNDC9, FOXA3, FPGS, FREM1, FRMD8, FSD1L, FUZ, FXR1, GABBR2, GABRQ, GATAD1, GDF10, GDF5, GDF6, GGA1, GGT5, GID8, GJA10, GKAP1, GMCL1, GNA12, GNAI3, GNB5, GNL1, GNLY, GORASP1, GORASP2, GPC5, GPN2, GPR15, GPRASP2, GRIK2, GRINA, GRP, GSPT2, GTF2F1, GTPBP2, GXYLT1, H3C10, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HIC2, HINT1, HLA-DOB, HLA-DQB2, HOXB9, HOXD3, HTR1A, HYLS1, IDS, IFNA10, IFNL3, IGFBP1, IGFBP4, IGHG1, IGHV7-81, IK, IL13RA1, IL17C, IL1R2, IL2RG, IMPA1, INKA2, INO80E, INPP5J, IQCG, IQUB, ITGB7, ITLN1, ITPA, JADE1, JAKMIP1, JAML, KCNAB2, KCND1, KCNMB1, KCTD10, KCTD13, KHDRBS3, KIAA0895, KIFC2, KLC2, KLHDC1, KLHL9, KLK10, KPTN, KRT79, KRTAP10-7, KRTAP4-4, L3MBTL4, LAMB3, LCN9, LDHA, LHX2, LIAS, LIMA1, LIMD1, LMNA, LMO2, LOXHD1, LPCAT2, LRFN5, LRP11, LRP3, LRRC18, LRRC45, LRRFIP2, LRTM1, LYPD1, LYPD5, LYPD6B, MACROD1, MACROH2A1, MAFF, MAGEB6, MAGEH1, MALT1, MANF, MAP6D1, MAPK15, MAPKAPK5, MAPRE3, MARCKS, MARS1, MAST2, MAST3, MAST4, MC3R, MCAM, MCM8, MCM9, MCU, MDGA2, METTL25, MGAT4B, MICU2, MIDN, MINDY1, MIPEP, MLST8, MMD, MMP11, MMUT, MORN1, MOXD1, MPND, MPP2, MPV17L2, MPZL1, MRAP2, MRM3, MRPL3, MRPL4, MRPL41, MRPL48, MRPL51, MRPS30, MRS2, MSMP, MSX1, MSX2, MTMR1, MTPAP, MUC3A, MVB12A, MX1, MXRA8, MYBL1, MYO1A, MYOC, MY0M3, NABP2, NAF1, NAGLU, NAIF1, NAXD, NCCRP1, NCDN, ND0R1, NDUFA7, NDUFS8, NELLI, NEUROG3, NFE2L3, NFIA, NFS1, NHLRC3, NINJ2, NIPAL3, NKAP, NMD3, NONO, NPDC1, NPNT, NR5A1, NRSN2, NTN5, NUBP1, NUDT3, NUDT9, NUP54, NUP62, NUP85, NXNL2, NXPE3, NYX, OAS1, ODF3, ODF3L2, OGGI, OLIG1, OLIG3, OPCML, OR10H1, OR4A15, OR4K17, OR51E1, OR5D14, OR8B12, ORC3, OSGIN1, OSTN, OTUB2, OTUD5, OXGR1, P2RY12, PACC1, PACSIN1, PACSIN2, PAK4, PARG, PARM1, PAX9, PCDH8, PCDHA10, PCDHB13, PCDHB2, PCSK7, PCYOX1L, PDE4A, PDE9A, PDHB, PDIA3, PDZD7, PEMT, PENK, PEX16, PFDN5, PFKFB3, PFKFB4, PFKL, PGRMC1, PHACTR3, PHF7, PICALM, PIGH, PIGP, PKM, PKP1, PLA2G3, PLAT, PLCG2, PLD6, PLEKHG5, PLEKHO2, PLP2, PLPPR2, PLXDC2, PMFBP1, PNMA2, PNOC, PNPT1, PGDN, POGLUT3, POLR3E, POLR3F, POU3F2, PPCDC, PPP1R12C, PPP2R1A, PPP2R2D, PPP3R2, PRDM1, PRKCD, PRLHR, PRMT2, PRNP, PRPF4, PRPF40A, PRR18, PRSS22, PSMA2, PSMD5, PTDSS1, PTGES2, PTK6, PTPN18, PTPRH, PTPRJ, PTPRS, PUM3, PWP1, PXMP4, PYROXD2, QPRT, RAB23, RAB39A, RAB42, RAB6B, RAD51B, RAFI, RALGPS1, RASSF2, RBM24, RBM3, RBM34, RBM46, RCC1L, RCN3, RDH10, RHBDD1, RHOH, RHOXF1, RILPL2, RIN1, RING1, RINL, RITA1, RNF126, RNF141, RNF144B, RNF220, RNF6, RO60, ROR1, RP2, RPH3AL, RPL30, RPL34, RPL6, RRS1, RSAD2, RTL8A, RUNDC1, RUNDC3A, RUSC1, RXFP3, SAFB2, SASS6, SBK1, SCG3, SCNN1A, SDF2L1, SDHAF2, SEC14L2, SEC61G, SEC63, SEL1L2, SEMA3G, SENP5, SERBP1, SERPINB5, SF1, SFRP1, SFT2D2, SFXN3, SGK1, SGO1, SH3GL2, SH3GLB1, SHH, SHKBP1, SHOC2, SIVA1, SKIL, SLC12A1, SLC1A7, SLC22A13, SLC22A7, SLC25A20, SLC25A43, SLC27A6, SLC2A13, SLC2A8, SLC37A3, SLC45A2, SLC49A4, SLF1, SMAD6, SMARCE1, SMG5, SNAP91, SNAPC2, SNRPB, SNRPG, SNX14, SNX27, SOWAHA, SPAG5, SPATS2, SPIRE1, SPRTN, SPTLC2, SRC, SRF, SSH3, SSX3, ST3GAL6, STARD7, STIM1, STK3, STK35, STMN1, STOML3, STX10, STXBP4, SUSD3, SUSD6, SYP, TAC1, TAFA5, TBC1D19, TBCK, TBL3, TBRG4, TCAF1, TCEA2, TCF19, TCP11, TCP11L1, TDG, TDP2, TEX13A, TGM4, TGOLN2, THAP11, THBD, THOC5, TIMM29, TLR2, TM4SF4, TMED6, TMEM106B, TMEM178B, TMEM204, TMEM234, TMEM263, TMEM41A, TMEM86A, TMIGD2, TMPRSS1 IE, TMPRSS2, TNFAIP1, TNFAIP8L1, TNFRSF10C, TNFSF13B, TOR1B, TOX2, TPD52L2, TPM4, TPO, TPSD1, TPT1, TRAPPC10, TRAPPC8, TRIM40, TRIM55, TRIR, TRMT12, TRPC5, TRPV2, TSKS, TTC12, TTLL7, TUBA1C, TUBGCP2, TXNDC5, UBAC1, UBXN2A, UGT3A1, UIMC1, UNCI 19, UQCRFS1, USH1C, USP15, USP21, VAC14, VEGFA, VPS37B, VPS45, VRTN, WAPL, WDR1, WDR24, WDR54, WDR5B, WDR60, WDR61, WIPI2, WNT2, XAF1, YBX2, ZAP70, ZBTB5, ZBTB46, ZC2HC1A, ZC3H3, ZCCHC8, ZDHHC1, ZDHHC13, ZFP2, ZFP36L2, ZGRF1, ZMPSTE24, ZNF175, ZNF19, ZNF205, ZNF274, ZNF428, ZNF436, ZNF502, ZNF558, ZNF624, ZNF668, ZNF71, ZNF710, ZNHIT2, and ZSWIM1. In some embodiments, the immunosuppressive resistance gene is ZBTB46.
[0044] In some embodiments, the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, ADAT1, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1, B9D2, BAB AMI, BAG5, BCHE, BLK, BMPR1B, BPIFC, BRF2, BSND, BTNL3, BYSL, C10orf82, C18orf25, C18orf54, Clorfll5, Clorf43, Clorf56, C1QTNF2, C1R, C2CD2, C5orfl5, C6orfl20, CAB, CA5B, CA8, CA9, CABS1, CALCOCO1, CALCR, CALHM3, CAMK2A, CAMLG, CARD8, CASP1, CASP7, CASTOR1, CBX7, CCDC110, CCDC69, CCDC82, CCDC84, CCL2, CCL21, CCR8, CCSER1, CD151, CD300LF, CD48, CD83, CD86, CDH7, CDK1, CDPF1, CDR2, CELF1, CEP63, CERS1, CES3, CFAP410, CGGBP1, CHAF1B, CHMP2A, CHMP7, CHRM1, CHST9, CISH, CLC, CLDN6, CLEC5A, CLIC5, CLP1, CLTRN, CLUAP1, CMTM7, CNTF, COG3, COL25A1, COL8A2, COMMD4, COPZ1, COPZ2, COQ4, COX6B2, CPLX2, CRACR2B, CROT, CRTAC1, CRY2, CSF1, CSNK1G2, CST9, CSTF3, CTDP1, CTNNA2, CTSF, CXCR6, CXXC1, CYP27A1, CYP2D6, CYTL1, DAXX, DBN1, DDHD2, DDX24, DES, DEXI, DGLUCY, DHX36, DNAI2, DNAJA2, DNAJB5, DNAJB6, DNAJC11, DNAJC27, DNAJC6, DOK5, DOK6, DRGX, DUS IL, DUSP5, DZIP1L, ECI2, EFCAB7, EGFL6, EGLN3, EIF3K, EIF4EBP1, ELSPBP1, EMC3, EPDR1, EPN1, EPO, ERFE, ERVK3-1, ESMI, F2RL2, FAAP100, FADS1, FAM172A, FAM53C, FAM71C, FAM81A, FBLN5, FBXL16, FBXO7, FBXW11, FCGR3A, FCRL5, FDFT1, FETUB, FEZF2, FGF10, FGF19, FGL1, FKBP5, FKBP9, FMOD, FNDC9, FOSB, FRMD3, FRMD5, FRZB, FUT3, GAB3, GABRA4, GABRG2, GALNT2, GALNTL6, GAS7, GBA2, GBP6, GEMIN8, GET1, GFM2, GFRA3, GK2, GLIPR1, GLRA2, GLRB, GLYCTK, GNA14, GNB3, GNL3, GOLM2, GPATCH2L, GPATCH3, GPM6B, GPR176, GPR45, GRAMD1B, GRB7, GRK2, GRK7, GSTM3, GXYLT1, GZMM, HAPLN3, HAUS2, HBG2, HCRTR1, HDAC8, HDGF, HEMK1, HERPUD1, HESX1, HEXA, HMHB1, H0XB5, H0XB6, H0XD3, H0XD4, HPGDS, HSD17B6, HSD17B8, HSPA2, HTN1, HTR5A, IFITM3, IFNA10, IGF1, IGFALS, IGHM, IL11RA, IL17A, IL17RE, IL2RB, IL12RB2, IL4, INSL6, ISL2, ISM2, IST1, ITPRID2, JAML, JUN, JUNB, KBTBD7, KCNA6, KCNN3, KEAP1, KIF3A, KIFC2, KIR2DL1, KLHDC2, KLHDC7B, KLHL9, KRT19, KRT6A, LARS2, LCK, LCN1, LDAH, LDLRAD4, LETMD1, LHX4, LHX9, LIN28A, LIPG, LNX1, LPAR5, LPL, LRFN3, LRRC15, LRRC2, LRRC34, LRRC42, LTBR, LYZ, MAF1, MAGOH, MAN1A1, MAN1B1, MAP2K6, MAP3K7, MAP4K5, MAPKAPK2, MAPKAPK5, MARCHF1, MARCHF2, MBNL1, MBP, MC3R, MCAM, MED26, MEIS3, METTL27, METTL2B, MIA2, MIF, MIPOL1, MLKL, MMD, MMP10, MOB4, MPHOSPH8, MPND, MPZL1, MR1, MRAS, MRM3, MRPL21, MRPS24, MRPS28, MS4A3, MS4A5, MSRA, MTA2, MTA3, MTHFD2, MTMR3, MTPAP, MTRF1L, MUS81, MYCL, MYCN, MYL10, MYL9, MYOC, MYOM3, MYORG, NAA80, NAPSA, NARF, NAT1, NCK1, NDE1, NDOR1, NDUFA13, NDUFA4, NEK5, NEEFE, NFIB, NFKBIB, NINJ2, NIPAL1, NKAIN2, NKAP, NMB, NOC4L, NPIPB15, NRARP, NRSN2, NTF3, NXNL2, OBP2A, OLR1, OR10AG1, OR10K2, OR14C36, OR1F1, OR2M3, OR2T8, OR5C1, OR7A5, OR9Q1, ORAI3, ORC2, ORM1, ORM2, OSGIN1, OSR2, OTUD5, P3H4, P4HA3, P4HTM, PAK2, PAK4, PBX2, PCDHA2, PCDHB12, PCDHGA2, PDYN, PFDN4, PFKP, PFN4, PGK1, PGLYRP1, PHF23, PHF7, PHKG1, PHOSPHO1, PI15, PI3, PI4KB, PITHD1, PKIA, PKNOX2, PLA2G3, PLA2G7, PLAUR, PLEKHA8, PLEKHO2, PLET1, PLOD2, PNPT1, POPDC3, PPM1D, PPME1, PPP1R2, PPP1R32, PPP2R2B, PPP2R3C, PPP2R5C, PPP6R2, PRKCB, PRKRIP1, PRKY, PRMT8, PRSS3, PRUNE2, PSCA, PSG1, PTGER3, PTK6, PTOV1, PTPRO, PTTG1IP, PUDP, PWWP3B, PYCARD, PYGB, QPCT, QPRT, RABI IB, RAB25, RAB28, RAB34, RAB40B, RABEP2, RADU, RAET1E, RAMP1, RARS1, RAX2, RBBP5, RCHY1, RCN3, REEP2, RFC4, RFPL2, RFX3, RIBCI, RINL, RIPK4, RLBP1, RNASE9, RNASET2, RNF111, RNF112, RNF144B, RNF24, RNF38, RNF7, ROPN1L, RP9, RPL6, RPS2, RPS3A, RPS6, RRAGA, RRAGD, RRP1, RRP9, RSAD1, RUBCN, RUNX1T1, RUVBL1, SAMHD1, SARS1, SCNN1A, SCNN1B, SCNN1G, SEL1L2, SELENBP1, SEPHS1, SEPTIN10, SEPTIN12, SERINC2, SERPINA3, SERPIND1, SERPINE1, SERPINE3, SETD3, SFRP4, SGK2, SH3KBP1, SHARPIN, SHISA3, SHOC2, SHOX, SIGLEC7, SIRPB2, SIRPG, SIRT3, SKIL, SLC13A1, SLC14A1, SLC20A2, SLC22A13, SLC22A31, SLC22A8, SLC25A1, SLC25A46, SLC25A47, SLC25A48, SLC2A8, SLC37A3, SLC39A7, SLC5A12, SLC6A19, SLC6A7, SLC7A9, SLCO1A2, SLFNL1, SMG9, SMPX, SNORC, SNRNP25, SNRPB, SNRPN, SNX16, SOAT1, SOCS5, SPATA22, SPATS2, SPC25, SPG21, SPINT1, SPINT2, SPNS2, SPP2, SQOR, SRC, SRFBP1, SRP54, SRP9, SRSF9, SSBP2, SSPN, STAP1, STARD7, STIM1, STK11, STX8, SULT4A1, SUMF2, SURF6, SYMPK, SYNCRIP, SZT2, TAS2R40, TAS2R60, TBCC, TBRG4, TBX20, TBX3, TC2N, TCEA1, TCEA2, TCF19, TCF7L2, TCN2, TCTN1, TDP2, TENT5C, TEX35, TFCP2L1, TFDP2, TGFB3, THAP12, TIAM2, TICAM2, TIPIN, TKT, TLE4, TM2D2, TM9SF3, TMEM106B, TMEM143, TMEM160, TMEM211, TMEM237, TMEM270, TMEM30A, TMEM39B, TMEM45A, TMEM68, TMPRSS3, TNFSF12, TOMM70, TOR1AIP1, TOX, TPP1, TPRKB, TPST2, TRAPPC12, TRDMT1, TRIM10, TRIM47, TRIM63, TRIP10, TRMO, TRMT44, TRPV4, TSN, TSPAN31, TSPEAR, TSSK3, TTBK2, TTC32, TUBA3C, TUBA3D, TUT7, TYW3, UBA1, UBASH3B, UBE2S, UBXN2A, UCHL3, UCHL5, UQCR10, USP1, USP19, VAT1L, VMA21, VPS29, VPS36, VSTM2A, VWA1, WAS, WDFY1, WDR4, WDR59, WDR63, WDR78, WNT11, WNT3A, WNT9A, YAF2, YJU2, ZBTB48, ZC3HAV1L, ZCCHC2, ZCCHC7, ZFP36L2, ZIC3, ZNF165, ZNF830, ZP2, ZSCAN21, and ZSCAN9. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL.
[0045] In some embodiments, the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of AB AT, ABHD12, ABH, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, ALOXE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GALNT2, B3GALT4, B3GNT2, BAB AMI, BAP1, BCAP31, BCCIP, BCL2L2, BCR, BDNF, BECN1, BEND2, BEND7, BLOC1S4, BMP5, BMPR1B, BRD3, BRF2, BRINP3, BTBD17, BTNL3, C10orf62, C12orf42, C14orf28, Clorf210, C2orf78, C6, C7orf31, CAB, CACNB3, CALR3, CARD14, CARD19, CASC1, CASP10, CASQ2, CBFB, CBX3, CBX4, CCDC141, CCDC148, CCDC68, CCDC8, CCDC82, CCIN, CCNG2, CCNY, CCR1, CCR10, CCR3, CD19, CD244, CD47, CD5L, CD83, CDC42EP1, CDC42EP4, CDCA2, CDCP2, CDH13, CDYL2, CEP120, CEP63, CERCAM, CERKL, CFAP100, CFAP20, CFAP410, CFAP47, CFAP91, CFHR1, CHGA, CHUK, CIRBP, CLCNKB, CLDND1, CLU, CMTR1, CNPPD1, COA3, COG1, COL22A1, COL25A1, COL6A2, COL8A2, COPB1, COPS3, COQ3, COQ8B, COX18, CPOX, CPQ, CPT1A, CPXM1, CRACR2A, CREB3L1, CRNN, CRTAP, CRYGD, CS, CSF1, CSF1R, CSF2RB, CSF3R, CSGALNACT1, CSTF2, CTCF, CTSV, CUEDC1, CUL2, CXADR, CYB5R2, CYP20A1, CYP26A1, CYP2C19, CYP8B1, DAAM2, DAB1, DACT2, DARS1, DBN1, DCAF4L2, DCAF8, DCT, DDO, DDR1, DDX43, DDX54, DEAF1, DECR1, DENR, DESI2, DHX40, DIP2A, DKK4, DNAAF3, DNAI2, DNAJC7, DNAL1, DPP4, DPT, DPYSL4, DRG1, DSCAM, DTL, DTX2, DUS1L, DUSP12, E2F7, ECHI, EFEMP2, EFHC1, EFS, EIF2B3, EIF4A2, EIF5, ELAVL4, ELL, ELMOD3, ELN, ELOA, ELOA2, ELP3, EMC7, ENCI, ENDOV, ENPP3, ENTPD5, EOLA2, EPB41L1, EPHB6, EPN1, ERCC6, ESRP1, ESRRA, ETFDH, EXO1, EXOC3, EXOSCIO, EXOSC8, EYS, F3, FABP6, FAM117A, FAM117B, FAM118B, FAM161B, FAM200A, FAM20A, FAM47A, FARSA, FBLN1, FBLN5, FBP1, FBXO24, FBXO40, FCHO1, FCRL5, FCRLB, FECH, FEM1B, FGF3, FGG, FKBP6, FLVCR1, FMNL1, FMO5, FNIP1, FOS, FOSB, FOXD4, FOXJ1, FOXM1, FOXN3, FOXRED2, FREM1, FRMD5, FRS2, FSTL1, FSTL4, FSTL5, FTMT, FXR2, G6PD, GABPB1, GABRB1, GALNT7, GANAB, GAPDHS, GATA2, GBP4, GCAT, GCGR, GCNT7, GCSAML, GDF2, GDF6, GET4, GGA1, GHDC, GINS4, GK, GKAP1, GLB1, GLRA2, GLRX5, GLYR1, GMCL2, GNA14, GNAT2, GNB3, GNL1, GNL3, GPAT4, GPC3, GPC4, GPC5, GPR84, GRHL3, GRHPR, GRIA4, GRID1, GRIK3, GRK4, GRM3, GRM8, GRN, GSDME, GSK3A, GSN, GTF2A1, GTF2B, GTF2E1, GUCY1B1, GYSI, HABP2, HADHA, HADHB, HDAC10, HEPHL1, HERPUD2, HES1, HEXD, HHIPL2, HINT2, HLA-C, HOMER3, HOOK3, HOXB6, HOXD3, HPS3, HPS5, HSD11B2, HSD17B13, HSD17B7, HSD3B1, HSPB9, HTR2B, IFNA6, IFNL1, IFT27, IGHA1, IGSF10, IGSF21, IL10RB, IL12RB2, IL13RA1, IL21, IL26, IL7R, ILVBL, IMPG1, INA, INHA, INSL4, INTU, IQUB, IRAG1, IRAKI, IREB2, IRF3, IRF5, IRX3, ITFG1, ITGB2, ITGB7, ITIH5, ITM2B, JAKMIP1, KANSL3, KAT5, KCNG3, KCNJ14, KCNK12, KCNK2, KCNK9, KCNMB3, KCTD12, KCTD4, KIAA2013, KIF2C, KIF3A, KIF3B, KIFC2, KITLG, KLC3, KLHDC2, KLHL13, KLHL21, KLHL8, KLK1, KRT6A, KXD1, L3MBTL4, LAD1, LAMP1, LAP3, LARS2, LDLRAD3, LGMN, LHX2, LHX4, LIMA1, LIMD1, LIPH, LIPT1, LKAAEAR1, LNPEP, LOXHD1, LOXL3, LTA4H, LTBR, LUC7L2, LYL1, LZTS2, MAG, MAN1A1, MANEA, MANF, MAP3K7, MAP4K5, MAP6D1, MAPK15, MAPK8, MAPKAPK5, MAPKBP1, MARS2, MATN2, MBLAC1, MCAM, MCOLN2, MCOLN3, MDH1B, MDM4, MEAK7, MECP2, MED1, MEOX1, METAP1, MFAP3L, MFNG, MITD1, MLEC, MLH1, MLLT3, MMP10, MMP16, MMS19, MNAT1, MON1B, MPP2, MPP7, MRM3, MRPL19, MRPL3, MRPL47, MSLN, MSRA, MTARC2, MTHFR, MTMR4, MTMR6, MUTYH, MVP, MYBL1, MYCN, MY01A, MYO5C, MYOC, MY0M3, MYORG, MYT1, NAGLU, NAMPT, NCKIPSD, NDC80, NDRG4, NDUFAF7, NDUFB6, NDUFS8, NDUFV2, NECTIN4, NELFA, NFIB, NIF3L1, NIPAL1, NIPBL, NMD3, NOC2L, NPLOC4, NPY, NPY2R, NQO1, NR2E1, NR6A1, NT5DC2, NTM, NTN5, NTNG1, NUDT8, NUP210, NXPE3, OAS1, OAS2, ODF2, OGA, OGFRL1, OLFM2, OLFML1, OLFML2B, OLIG1, OLR1, OMP, OR4D1, OR4S2, OR52N5, OR52W1, OR5B3, OR9Q1, ORMDL2, OSBPLIO, OSGEPL1, OTX1, OXSR1, P2RX6, P2RY1, PACS2, PAEP, PAFAH1B2, PAK4, PARL, PARP9, PARS2, PC, PCDHA1, PCDHA6, PCDHGA2, PCDHGA5, PCSK2, PDCD6, PDE4A, PDIA3, PDK2, PDZD9, PENK, PFKL, PGAM5, PGK1, PGM2, PHF11, PHYH, PICALM, PIK3R1, PIP5K1B, PLAGL1, PLCD4, PLD1, PLEK, PLEKHG5, PLEKHS1, PLIN1, PLK1, PLPPR2, PM20D1, PNMA8A, POLE, POLI, POLR2K, POLR3F, POU3F2, PPA2, PPIG, PPM1D, PPP1R12C, PPP1R16B, PPP1R32, PPP2R3C, PPP2R5C, PPP2R5D, PRAC1, PRAM1, PRAME, PRDM1, PRDX3, PRKAA2, PRKAG2, PRKAR1B, PRKCE, PRMT1, PROC, PRRT2, PRSS45P, PRSS48, PSAPL1, PSD3, PSEN1, PSMC4, PSMD14, PTBP3, PTCD2, PTDSS1, PTGER2, PTK6, PTPN12, PTPRN, PUF60, PUM1, PYDC1, PYGB, RAB1A, RAB33B, RAB42, RABL6, RAD18, RAD51, RAET1G, RALBP1, RASSF4, RBBP7, RBM12, RBM38, RBM4, RBM46, RBM4B, RBX1, RCBTB2, RCC1L, RDH10, RDH12, REC8, REN, RET, RFC2, RFC5, RFX4, RGS16, RHAG, RHEX, RHOBTB2, RICTOR, RIMBP2, RIMS3, RIOK3, RIPK1, RIPK2, RIT1, RMDN3, RNASEH2B, RNF114, RNF148, RNF213, RNF6, RNPEPL1, RO60, RORA, RPL30, RPS14, RPS4X, RPS6KA2, RPS6KB1, RSRC1, RTF1, RTN1, RUBCN, RUNDC1, RUNX3, S100PBP, SAMHD1, SCAMP2, SCIN, SCNN1D, SCRN2, SDSL, SEC63, SEL1L3, SELL, SENP3, SEPTIN10, SEPTIN8, SERINC3, SERPINF1, SGK3, SGMS1, SH3GLB1, SHOC2, SIAH2, SIGLEC10, SIGLEC12, SIGLEC7, SIRPG, SIRT5, SKAP1, SKIL, SLC15A3, SLC16A1, SLC16A7, SLC18A2, SLC1A7, SLC20A2, SLC22A2, SLC22A23, SLC22A24, SLC23A2, SLC25A19, SLC25A25, SLC25A47, SLC26A2, SLC2A4, SLC2A8, SLC36A3, SLC37A2, SLC37A3, SLC39A14, SLC43A1, SLC5A11, SLC5A12, SLC5A7, SLC6A7, SLC7A1, SLCO1A2, SLITRK3, SMARCD3, SMOX, SNCAIP, SNTA1, SOCS6, SOX9, SPATA2, SPCS3, SPN, SPNS2, SPOCK3, SPOUT1, SPRR4, SRC, SRD5A3, SRFBP1, SRMS, SSMEM1, SSX2IP, ST7L, STEAP1, STIM1, STK11, STK17A, STK17B, STK3, STK39, STXBP1, STXBP2, STXBP3, STXBP5, SUGCT, SUN5, SUSD2, SYNCRIP, TAB2, TBC1D10A, TBC1D22B, TBC1D9B, TBCD, TBL1X, TBX6, TON, TCF25, TCTN2, TDGF1, TEAD2, TESK1, TESK2, TEX48, TFAP2A, TFAP4, TGFB1, TGFBI, TGOLN2, THAP3, THAP7, THEM4, THOC1, THOC7, TH0P1, THSD4, TIAM2, TICAM2, TLE6, TLK1, TM9SF4, TMED2, TMEM259, TMEM62, TMIE, TMLHE, TM0D1, TMTC3, TNF, TNS1, TOMM70, TRAF3IP1, TRAM1, TRAP1, TRAPPC3, TRAPPC4, TRAPPC8, TRAPPC9, TRIB3, TRIM32, TRMT13, TRPM3, TRPV4, TSGA10, TSPEAR, TSPYL6, TTC13, TTC16, TTC26, TTC38, TTC8, TTF2, TTLL2, TTLL7, TUBA3E, TUBA8, TUBB3, TUBB4B, TUFM, TYMS, UBE2O, UBE2Z, UBE3A, UBE3C, UBOX5, UGDH, UGP2, UGT3A1, UGT8, UMOD, USP1, USP49, UXS1, VANGL2, VASN, VEGFA, VIL1, VPS45, VWA1, WAPL, WDR37, WDR63, WDR90, WDR91, WNT4, WWOX, XRCC5, YBX2, YME1L1, ZBTB14, ZBTB18, ZBTB46, ZC3H10, ZC3H11A, ZCCHC8, ZDHHC11, ZDHHC15, ZDHHC6, ZFYVE21, ZGRF1, ZMAT3, ZNF165, ZNF18, ZNF19, ZNF20, ZNF224, ZNF25, ZNF277, ZNF334, ZNF350, ZNF354C, ZNF396, ZNF398, ZNF436, ZNF461, ZNF467, ZNF496, ZNF560, ZNF565, ZNF566, ZNF597, ZNF610, ZNF623, ZNF668, ZNF74, ZP1, ZPLD1, ZSCAN25, and ZSWIM2. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of CD47, CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZTBTB46.
[0046] In other aspects, provided herein is a method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of: (i) collecting peripheral blood mononuclear cells (PBMCs) from an individual, (ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i), (iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and (iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
[0047] In some embodiments, the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR). In some embodiments, the exogenous nucleic acid further comprises a T cell receptor (TCR).
[0048] In some embodiments, the peripheral blood mononuclear cells are obtained from leukapheresis.
[0049] In some embodiments, the CD8+ and CD4+ T cells are isolated sequentially. [0050] In some embodiments, the naive CD4+ T cells are differentiated into activated CD4+ T cells. In some embodiments, the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell). In some embodiments, the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid. [0051] In some embodiments, the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element. [0052] In some embodiments, the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
[0053] In some embodiments, the transduced lymphocytes are enriched by positive selection. In some embodiments, the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
[0054] In other aspects, provided herein is a method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising: (i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual, (ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes, (iii) transiently stimulating the transduced lymphocytes, (iv) exposing the transduced lymphocytes to an immunosuppressive environment, (iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and (v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene. In some embodiments, the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. [0056] FIGs. 1A-1B shows the dose-dependent suppression of T cell proliferation by Adenosine. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti- CD3/CD28 antibodies and cultured in the presence of increasing concentrations of adenosine for 4 days. FIG. 1A quantifies the suppression by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of adenosine), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions. FIG. IB shows the viability of T cells following 4 days of culture in the presence of adenosine. [0057] FIGs. 2A-2B shows the dose-dependent suppression of T cell proliferation by TGF-p. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of increasing concentrations of TGF-P for 4 days. FIG. 2A quantifies the suppression by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of TGF-P), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions. FIG. 2B shows the viability of T cells after 4 day culture in the presence of TGF-p.
[0058] FIGs. 3A-3E shows the dose-dependent suppression of T cell proliferation by coculture with nTregs and iTregs. After 9 days post-isolation, iTregs (FIG. 3A) and nTregs (FIG. 3B) were stained for expression of the transcription factor FoxP3. Grey histograms are isotype controls while blue histograms are cells stained with anti-FoxP3 antibody. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of increasing number of iTregs (FIG. 3C) or nTregs (FIG. 3D) for 4 days. Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of Tregs), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions. FIG. 3E shows the ex vivo expansion of iTreg and nTreg cells after isolation.
[0059] FIGs. 4A-4B displays the suppressive effect of polarized macrophages on T cell proliferation. FIG. 4A shows representative staining of markers of immunosuppressive macrophage polarization. FIG. 4B depicts the percent suppression of T cells cultured in the presence of macrophages. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of equal numbers of macrophages, polarized for 4 days with different combinations of the following factors: dexamethasone, IL-4, IL-6, TGF-|31, TGF-|32, TGF-|33, IL-10, IL-ip. Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of macrophages), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
[0060] FIGs. 5A-5B shows the correlation of the percentage of proliferating T cells with their proliferation index (FIG. 5A) and absolute cell count (FIG. 5B). Gating on the most proliferating cells (the cells with highest dilution of CTY) correlates well with gold standard measures of T cell proliferation. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured with or without immunosuppression for 4 days. Proliferation index, the total number of divisions divided by the number of cells that went into division, was determined based on CTY dilution. Absolute cell count was measured by inclusion of absolute counting beads in the assay.
[0061] FIG. 6 shows a schematic representation of the immunosuppressive resistance gene screen in the context of TME immunosuppression.
[0062] FIGs. 7A-7B shows the effect of T cell stimulation on the ability to suppress T cells ex vivo. Primary CD4+ and CD8+ T cells were labeled with CTY, stimulated with anti- CD3/CD28 antibodies and cultured in the presence of different immunosuppressive factors and cell types. Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of immunosuppression), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions. The activator (anti-CD3/CD28 antibodies) was either present throughout the 4- day duration of the assay or removed after 6 h, prior to co-incubation of T cells with immunosuppressive factors (FIG. 7A). FIG. 7B shows the percent suppression of T cell proliferation obtained using a 400 lU/mL or 50 lU/mL concentration of IL-2 during the 4-day duration of the assay. Nature 2022 indicates the methods used in the Legut et al., 2022 publication.
[0063] FIGs. 8A-8B depicts the quantification of screen quality metrics including coverage and cell number following T cell expansion (MM). Modifications to the immunosuppressive resistance gene screen enable multiple high quality immunosuppressive resistance gene screens from the same donor. FIG. 8A depicts the average number of transduced T cells per barcode in the immunosuppressive resistance gene library. FIG. 8B shows the total number of viable T cells after a 14-day expansion. Nature 2022 indicates the data published in Legut et al., 2022 publication.
[0064] FIG. 9 depicts the level of immunosuppression achieved in the context of the immunosuppressive resistance gene screens. Primary CD4+ and CD8+ T cells from three healthy donors, transduced with the immunosuppressive resistance gene library, were labeled with CTY, stimulated with anti-CD3/CD28 antibodies and cultured in the presence of different immunosuppressive factors and cell types. Suppression was quantified by comparing the frequency of top proliferating T cells in each condition to control T cells (T cells cultured in absence of immunosuppression), gating on the 20% most proliferating control T cells and applying the same gate to all the conditions.
[0065] FIG. 10 shows the quantification of barcode recovery in each immunosuppressive resistance gene screen. Modifications to the immunosuppressive resistance gene screen enable improved data quality. Following sorting of the most proliferating T cells, the presence and relative abundance of each barcode present in the library was quantified by sequencing. Nature 2022 indicates the data published in Legut et al., 2022 publication.
[0066] FIG. 11 shows that overexpression of representative immunosuppressive resistance genes enhances CAR-T cell killing in the presence of regulatory T cell (Treg) immunosuppression in vitro. CD4+ and CD8+ T cells co-expressing a HER-2- specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
[0067] FIG. 12 shows that overexpression of representative immunosuppressive resistance genes enhance CAR-T cell killing in the presence of adenosine immunosuppression in vitro. CD4+ and CD8+ T cells co-expressing a HER2-specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
[0068] FIG. 13 shows that overexpression of representative immunosuppressive resistance genes enhance CAR-T cell killing in the presence of macrophage (mac) immunosuppression in vitro. CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2- 28z CAR) and an indicated immunosuppressive resistance gene were co-incubated with GFP- engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). Mean and SEM are shown.
[0069] FIGs. 14A-14B show that overexpression of the immunosuppressive resistance gene ZBTB46 enhances CAR T cell killing of cancer cells in the presence of immunosuppression and alleviates T cell exhaustion in vitro. CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and ZBTB46 or a control irrelevant gene were co- incubated with GFP-engineered HER2+ cancer cell lines at 1:4 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to account for the presence of a specified immunosuppressive factor on cancer cell growth. Mean and SEM are shown. CAR T cells were co-incubated with cancer cells and were challenged with fresh cancer cells every 3-4 days. The assay was conducted in culture medium without (FIG. 14A) or with exogenous TGF-p (FIG. 14B).
[0070] FIG. 15 shows that overexpression of the immunosuppressive resistance gene LTBR results in alleviation of CAR-T cell exhaustion and enhanced cancer cell killing capacity in a repeated cancer challenge assay compared to benchmark CAR-T armoring genes (cJUN, mblL15, and TGFBR2dn) in vitro. CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and LTBR were co-incubated with GFP-engineered HER2+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. When CAR T cells stopped specifically killing cancer cells, they were removed from the subsequent rounds of cancer cell challenge. Mean and SEM are shown.
[0071] FIG. 16 shows that overexpression of the immunosuppressive resistance gene LTBR, but not the benchmark CAR T armoring genes, enhances cancer cell killing in the presence of immunosuppressive factors in vitro. CD4+ and CD8+ T cells (aP T cells) co-expressing a HER2-specific CAR (HER2-28z CAR) and indicated gene were co-incubated with autologous regulatory T cells (Treg) and GFP-engineered HER2+ cancer cell lines at 1 : 1 T cells to cancer cells ratio. After 4 days, T cells were challenged with fresh cancer cells as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
[0072] FIG. 17 shows that overexpression of the immunosuppressive resistance gene LTBR enhances CAR T cell killing of CLDN6+ expressing ovarian cancer cell lines PAI and OV90 in vitro. CD4+ and CD8+ T cells (aP T cells) co-expressing a CLDN6-specific CAR (CLDN6-BBz CAR) and LTBR or a control irrelevant gene were co-incubated with GFP- engineered CLDN6+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells, as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
[0073] FIGs. 18A-18C show that overexpression of the immunosuppressive resistance gene LTBR enhances cancer killing and T cells expansion while reducing T cell exhaustion of CAR gamma delta T cells in vitro. Gamma delta T cells co-expressing a CLDN6-specific CAR (CLDN6-BBz CAR) and LTBR or a control irrelevant gene were co-incubated with 1 GFP-engineered CLDN6+ cancer cell line OV90. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. After 96 h of coincubation, T cells were harvested and stained in the presence of counting beads to determine their expansion and exhaustion. Mean and SEM are shown. FIG. 18A shows percent of OV90 killing by CAR-T cells at 72 h of co-incubation at 1:32 T cell to cancer cell ratio. FIG. 18B shows the fold-expansion of CAR gamma delta T cells with or without LTBR overexpression following co-incubation with OV90 cancer cells. FIG. 18C analyzes the percentage of exhausted phenotypic CAR gamma delta T cells following co-incubation with OV90 cancer cells. Exhausted cells are defined as LAG3+ TIM3+.
[0074] FIG. 19 shows that overexpression of the immunosuppressive resistance gene LTBR enhances killing of antigen-low cancer cell line by CAR gamma delta T cells in vitro. Gamma delta T cells co-expressing a CLDN6-specific CAR (CLDN6-28z CAR) and LTBR or a control irrelevant gene were co-incubated with GFP-engineered CLDN6low cancer cell line SKOV3 for 88 h at 1:4 T cell to cancer cell ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Mean and SEM are shown.
[0075] FIGs. 20A-20D show that LTBR enhances CLDN6-28z CAR efficacy in an animal model of ovarian cancer without causing apparent toxicities. FIG. 20A shows the effect CAR T cells co-expressing a control gene tEGFR (n = 4) or LTBR (n =5) on tumor burden compared to donor-matched T cells not expressing any CAR (“No CAR”, n=13). Here, NSG mice were injected i.v. with 1.25 x 106 CAR T cells co-expressing a control gene tEGFR (n = 4) or LTBR (n =5), or donor-matched T cells not expressing any CAR (“No CAR”, n=13). Following T cell injection, survival of the animals was monitored daily. Animals were removed from the study and euthanized upon reaching a tumor burden of over 2,000 mm3 or another humane endpoint. * p<0.05 (log-rank test). FIG. 20B shows body weight measurement since T cell injection, normalized to starting body weight. Dotted line represents a humane endpoint of 20% body weight loss. FIG. 20C shows an assessment of tumor infiltration by human T cells at the experimental endpoint. At the experimental endpoint, matched tumor samples were explanted and processed to assess tumor infiltration by human T cells. Data normalized to the number of T cells detected in the tumor explanted from mice treated with CAR + tEGFR. FIG. 20D shows relative CLDN6 levels as determined by flow cytometry comparing the staining on tumor cells freshly explanted from animals to OV90 cell line cultured in vitro and processed in parallel. [0076] FIG. 21 shows enhanced CLDN6-28z y5 CAR efficacy with LTBR expression in a repeated challenge model in vitro. CAR T cells, co-expressing a control gene tNGFR (“Unmodified CAR”) or armoring genes LTBR, mbIL15 or TGFpRIIDN were co-incubated with GFP+ OV90 ovarian cancer cells at 1:1 effector:target ratio for 72-96 hours. After each round of co-incubation, T cells were harvested and added to fresh cancer cells. Killing was determined at endpoint of each co-incubation by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone.
[0077] FIG. 22 shows enhanced CLDN6-28z aP CAR efficacy with LTBR expression in a tumor cell killing assay in vitro. CAR T cells, co-expressing a control gene tEGFR (“Unmodified CAR”), armoring gene LTBR or a truncated version of LTBR lacking the intracellular signaling domain (“LTBR del”) were co-incubated with GFP+ OV90 ovarian cancer cells at 1:32 effector: target ratio for up to 110 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone. [0078] FIG. 23 shows enhanced CLDN6 « CAR T cell efficacy with LTBR expression in a tumor cell killing assay in vitro. CAR T cells, co-expressing a control gene tEGFR (“Unmodified CAR”) or armoring gene LTBR were co-incubated with GFP+ SKOV3 ovarian cancer cells at 1:16 effector: target ratio for up to 140 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone.
DETAILED DESCRIPTION
[0079] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
[0080] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. DEFINITIONS
[0081] As used in this specification and the appended claims, the singular forms “a”, “an' and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.
[0082] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
[0083] Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; Band C; A (alone); B (alone); and C (alone).
[0084] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991): Qhtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolim et af., Mol. Cell. Probes 8:91-98 (1994)). [0085] The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.
[0086] Nucleic acids described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding aspects of a CRISPR-Cas editing system described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject. In some embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
[0087] ‘ ‘Variants” of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g., its specific inhibitory property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e., nonmutated physiological, sequence. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bonds, e.g., side chains which have a hydroxyl function. This means that e.g., an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g., using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modem Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam). A variant may also include a non-natural amino acid. [0088] A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75, 100 or more amino acids of such protein or peptide, or over the full length of the protein or peptide.
[0089] The term “gene” can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
[0090] As used herein, the terms “coding region” and “region encoding” and grammatical variants thereof, refer to an open reading frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.
[0091] “Polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
[0092] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. [0093] Unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. A nucleic acid sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
[0094] The term “expression” is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. Expression may be transient or may be stable.
[0095] The terms “expressing”, and “overexpression” refer to increasing the expression of a gene or protein. The terms refer to an increase in expression, for example, an increase in the amount of mRNA or protein expressed in a T cell, other lymphocyte or host cell, of at least 10%, as compared to a reference control level, or an increase of least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300% or at least about 400%. In some embodiments, the reference control level is the amount of mRNA or protein expressed in a cell that has not been transduced with nucleic acid encoding the protein. Various methods for expression and/or overexpression are known to those of skill in the art, and include, but are not limited to, stably or transiently introducing a heterologous polynucleotide encoding a protein (i.e., an immunosuppressive resistance gene set forth in Tables 1-6) to be expressed and/or overexpressed in the cell or inducing expression or overexpression of an endogenous gene encoding the protein in the cell. It is understood that one or more genes set forth in Tables 1-6 can be expressed and/or overexpressed in a cell. It is also understood that two or more genes to be expressed and/or overexpressed in a cell can be selected from one or more of the genes set forth in Tables 1-6.
[0096] The term “autologous” refer to any material derived from the same subject to whom it is later to be re-introduced.
[0097] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system. In some embodiments, an exogenous nucleic acid includes an additional copy of a nucleic acid sequence already existing in the organism, cell, tissue, or system. For example, an exogenous nucleic acid includes vectors comprising nucleic acid encoding a gene that is already present at its endogenous location in the cell.
[0098] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
[0099] As used herein, an “expression cassette” refers to a nucleic acid molecule which encodes one or more ORFs or genes, e.g., an immunosuppressive resistance gene, or a CAR or TCR or component thereof. An expression cassette also contains a promoter and may contain additional regulatory elements that control expression of one or more elements of a gene editing system in a host cell. In one embodiment, the expression cassette may be packaged into the capsid of a viral vector (e.g., a viral particle). In one embodiment, such an expression cassette for generating a viral vector as described herein is flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
[0100] The term “regulatory element” or “regulatory sequence” refers to expression control sequences which are contiguous with the nucleic acid sequence of interest and expression control sequences that act in trans or at a distance to control the nucleic acid sequence of interest. As described herein, regulatory elements comprise but are not limited to: promoter; enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and poly adenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Also, see Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleic acid sequence in many types of target cell and those which direct expression of the nucleic acid sequence only in certain target cells (e.g., tissue-specific regulatory sequences).
[0101] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The term “constitutive” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. The term “inducible” or “regulatable” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. In some embodiments, the inducible promoter is activated in response to T cell stimulation. In some embodiments, the promoter is heterologous. In some embodiments, the promoter is an NF AT, API, NFKB, or IRF4 promoter. The term “tissue- specific” when referring to a promoter specifies a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
Exemplary promoters include the CMV IE gene, EF-la., ubiquitin C, 5’LTR, or phosphoglycerokinase (PGK) promoters.
[0102] The term “operably linked” or refers to functional linkage between one or more regulatory sequences and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein coding regions, are in the same reading frame.
[0103] The term “lentivirus” refers to a genus of the Retroviridae family. Eentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
[0104] In some embodiments, one or more genes are encoded by a nucleic acid sequence that is delivered to a host cell by a vector or a viral vector, of which many are known and available in the art. In one embodiment, provided is a vector comprising an expression cassette as described herein. In one embodiment, a vector is a non- viral vector. In another embodiment, a vector is a viral vector. A “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of interest is packaged in a viral capsid or envelope. Examples of viral vectors include but are not limited to lentivirus, adenoviruses, retroviruses (y- retroviruses and lentiviruses), poxviruses, adeno- associated viruses (AAVs), baculoviruses, herpes simplex viruses. In one embodiment, the viral vector is replication defective. A “replication-defective virus” refers to a viral vector, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient, i.e., they cannot generate progeny virions but retain the ability to infect cells.
[0105] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
[0106] In some embodiments, the vector is a non-viral plasmid that comprises an expression cassette described herein, e.g., naked DNA, naked plasmid DNA, RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.
[0107] Plasmids, other cloning and expression vectors, properties thereof, and constructing/manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art. In one embodiment, an expression cassette as described herein is engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for introduction to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
[0108] RNA or DNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-11 (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
II. OVERVIEW
[0109] Provided herein are modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein. The modified lymphocytes disclosed herein display significantly improved proliferation when exposed to immunosuppressive cellular environments mimicking an immunosuppressive tumor microenvironment (TME). The immunosuppressive resistance genes disclosed herein comprise both immune response modifiers as well as genes not typically expressed by peripheral T-cells. The modified lymphocytes expressing the immunosuppressive resistance genes disclosed herein therefore address an important limitation currently impeding the establishment of effective cellular therapies for tumors comprising immunosuppressive microenvironments .
IL MODIFIED LYMPHOCYTES
[0110] Provided herein, in some embodiments, are modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
[0111] In some embodiments, the immunosuppressive resistance gene, when expressed in a lymphocyte, enables the lymphocyte to proliferate despite exposure to an immunosuppressive cellular environment. In some embodiments the immunosuppressive cellular environment is selected from selected from the group consisting of adenosine driven immunosuppression, TGF-P driven immunosuppression, regulatory T cell (Treg) driven immunosuppression, and macrophage driven immunosuppression.
[0112] In some embodiments, the immunosuppressive mechanism underlying adenosine driven immunosuppression comprises the adenosinergic pathway. In some embodiments, activation of the adenosinergic pathway occurs within hypoxic tumors. In some embodiments, the adenosinergic pathway comprises ectonucleotidases (CD39 and CD73) and adenosine receptors (AIR, A2AR, A2BR and A3R) that participate in the generation and signalling of adenosine in the tumour microenvironment (TME). In some embodiments, the cyclic AMP (cAMP)-activating receptors A2AR and A2BR predominantly exert immunosuppressive functions in the TME. In some embodiments, extracellular adenosine exerts local suppression through tumor-intrinsic and host-mediated mechanisms.
[0113] In some embodiments, the mechanism underlying TGF-P driven immunosuppression comprises inhibition of T cell proliferation. In some embodiments, exposure to TGF-P inhibits the IL-2 expression and secretion in T cells. In some embodiments, exposure to TGF- P inhibits expression of the Ifiig, Gzma, Gzmb, Prfl and Faslg genes in T cells. In some embodiments, exposure to TGF-P downregulates the expression of MHC molecules on the surface of tumor cells.
[0114] In some embodiments, Treg cells directly suppress anticancer immunity, thereby hampering effective anti-tumor immune responses in tumor-bearing individuals. In some embodiments, Tregs alter immune function in cells of both the innate and adaptive immune systems. In some embodiments, Tregs secrete immunosuppressive cytokines such as TGF-P, IL-10, and IL-35. In some embodiments, Tregs inhibit the metabolic functions of immune cells through CD25 (IL-2 receptor alpha) dependent cytokine deprivation facilitated apoptosis, immunosuppressive adenosine by ectoenzymes CD39 and CD73 and c-AMP mediated inhibition.
[0115] In some embodiments, the mechanism underlying macrophage driven immunosuppression comprises the activity of Tumor-Associated Macrophages (TAMs). In some embodiments, TAMs express inhibitory cytokines that dampen immune responses. In some embodiments, macrophages produce oxygen radicals that generate toxic compounds, including peroxynitrite and hydrogen peroxide, that suppress the proliferation and activity of immune cells. In some embodiments, macrophages directly suppress T cell activity and proliferation. A. Immunosuppressive Resistance Genes
[0116] In some embodiments, provided herein are modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene set forth in Tables 1- 6. In some embodiments, the immunosuppressive resistance gene is endogenously expressed by the lymphocyte. In some embodiments, the immunosuppressive resistance gene is not naturally expressed by the lymphocyte. In some embodiments, the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by at least about 10% more as compared to the amount of mRNA of the immunosuppressive resistance gene expressed in a non-modified lymphocyte. In some embodiments, the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by between about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% about 90% to about 100% more as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte. In some embodiments, the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by at least about 1.2-fold as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte. In some embodiments, the amount of mRNA of the immunosuppressive resistance gene is increased in the modified lymphocyte by about 1.2-fold, about 1.5-fold, about 1.7-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, or about 10-fold as compared to the amount of mRNA of the immunosuppressive resistance gene in a non-modified lymphocyte. [0117] In one aspect, this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 1, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. In some embodiments, increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking an adenosine- driven tumor microenvironment compared to an unmodified lymphocyte. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, AD ARBI, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP6V1C1, ATP6V1D, ATPAF1, ATRAID, ATRIP, AUTS2, AZI2, B3GNT9, BAIAP2, BAIAP2L2, BAP1, BATF, BCAT1, BCKDHA, BCL2L13, BCL2L2, BLMH, BMPER, BOD1, BPIFA1, BSG, BSPRY, BTC, BTD, BTRC, C18orf54, C1QTNF12, C1QTNF4, C3orf38, C6orf223, C8orf76, C9orfll6, CALCOCO1, CALHM1, CAT, CAVIN1, CAVIN4, CBWD1, CCDC113, CCDC121, CCDC14, CCDC174, CCDC86, CCDC93, CCIN, CCN2, CCN3, CCR6, CCSAP, CCT7, CD19, CD1B, CD36, CD46, CD86, CD96, CDC14A, CDH1, CDK2AP1, CDK7, CELA2B, CEP97, CFAP46, CFAP47, CHAMP1, CHMP2B, CHST8, CKB, CLEC11A, CLEC4M, CLEC9A, CLPB, CLPSL1, CLSTN1, CLUL1, CLYBL, CNDP2, CNTROB, COA6, COL15A1, COL21A1, COL22A1, COLQ, COMMD5, COPZ2, CORO6, COX11, CPA1, CPSF6, CPT2, CRB3, CREB3L2, CREM, CRHR2, CRTC2, CRYGD, CSK, CTBP2, CTNNA2, CTSS, CX3CR1, CYP20A1, CYP2C9, DADI, DAXX, DBF4, DCDC2, DCLK2, DCTN3, DCX, DDB1, DDHD1, DDO, DDOST, DDX21, DDX56, DEGS2, DEPDC7, DFFB, DIXDC1, DLK1, DNAI2, DNAJC16, DPPA4, DPYSL3, DPYSL4, DRG2, DUT, E2F7, EBNA1BP2, ECHDC1, ECHDC3, ECU, ECRG4, EDC3, EFCAB1, EFCAB12, EFCAB13, EFEMP2, EGR2, EIF2A, EIF4G3, EMP1, ENPP3, EPHA4, EPHB6, EPX, ERCC2, ERN2, ERV3-1, ESRP1, ETNK2, EVI2B, EXO1, EXTL3, EZH2, F10, F13B, F2, FAF2, FAM110A, FAM13C, FAM161B, FAM181A, FAM200A, FAM71A, FAM98A, FANCB, FATE1, FBXL13, FBXL16, FBXL20, FCRLB, FEM1C, FEV, FGF22, FGF3, FKBP14, FKBP5, FKBPL, FLOT1, FLVCR1, FLYWCH1, FMN1, FMO5, FOSB, FOXJ1, FOXS1, FPGT, FRMD5, FUT8, G6PC3, GAB3, GABRA5, GABRB2, GABRQ, GAD2, GALNT7, GAN, GAPDHS, GAPVD1, GBP4, GCNA, GDF6, GDF9, GDPD5, GET1, GET4, GGA1, GGA2, GGTLC1, GIMAP8, GKAP1, GLRB, GLT6D1, GLTPD2, GNAI2, GORASP1, GORASP2, GOSR1, GPAA1, GPC5, GPR119, GPR37, GPR65, GPRASP2, GPRC5C, GPT2, GPX1, GRHL2, GRID1, GRINA, GSDME, GTF2H1, GTF2I, GTPBP2, GUSB, GYPA, H2BC4, HABP4, HACD3, HAGH, HAND2, HARS2, HAUS7, HAX1, HDAC9, HDHD3, HENMT1, HEPACAM2, HHEX, HIBCH, HID1, HK1, HLA-C, HLCS, HNF4A, HNRNPAB, HNRNPD, HOXA11, HOXD8, HP1BP3, HPS3, HPS4, HSDL1, HSP90B1, HTR1F, HTRA4, HVCN1, IBSP, IDS, IFNA8, IFNAR2, IFNGR1, IFNLR1, IGFBP1, IGKV1D-17, IHO1, IK, IL10RA, IL12RB2, IL13RA2, IL17RE, IL36G, IMPDH2, INF2, INKAI, INTS5, IP6K2, IPO5, IQCA1, IREB2, JAGN1, JMJD8, KAT14, KAT7, KAZALD1, KCNAB3, KCNG3, KCNJ14, KCNK12, KCNK4, KCNK9, KCNMB2, KCNV1, KCTD4, KHDRBS2, KHDRBS3, KIAA0930, KIF7, KIFC3, KLHL14, KLHL2, KLHL8, KREMEN2, KRT13, KRT79, LARS2, LCK, LECT2, LGALS12, LHX4, LMX1B, LNX1, L0XL1, LRP3, LRP5L, LRRC18, LRRC45, LRRC55, LRRC61, LRRN3, LTBR, LYVE1, MAFB, MAG, MAGT1, MANSC1, MAP1LC3C, MAP2K1, MAP2K4, MAP2K5, MAP6D1, MAPK14, MARVELD2, MCAM, MCF2L, MCM10, MCOLN3, MED1, MEMO1, METTL14, MEX3B, MFAP4, MGAT4B, MGME1, MICU2, MIER3, MKS1, MLLT11, MLST8, MMP13, MOBP, MOVIO, MRPL3, MRPS27, MSTN, MTG2, MTHFD2, MTMR12, MTMR8, MYBL1, MYCL, MYO1A, MYORG, NACC2, NADSYN1, NAE1, NBPF15, NCAM1, NCDN, NCLN, NDUFA2, NDUFB7, NECAP1, NECAP2, NECTIN1, NECTIN2, NEK3, NF2, NFIL3, NGFR, NIBAN1, NID2, NIF3L1, NIPSNAP3B, NKX2-8, NOA1, NOXI, NPLOC4, NPNT, NR1I2, NRM, NRTN, NT5DC1, NTPCR, NUP37, NXF3, NXPE3, NXT1, OAT, OIT3, OLFM2, OR1F1, OR1N1, OR52E8, OR9A4, ORM2, OTUD7B, OTX1, OXCT2, P2RX4, P2RX5, P2RX7, P2RY6, P2RY8, P4HTM, PABPC3, PAF1, PAIP2, PARD6B, PARVA, PAX9, PCCA, PCDHGB1, PCLO, PDE1A, PDE4A, PDE9A, PDGFRA, PDHB, PDSS1, PDZD3, PDZD9, PELO, PFKL, PFKP, PI3, PI4K2A, PIGO, PIM1, PIMREG, PLA2G7, PLAGL1, PLAT, PLEKHO2, PLOD1, PLOD2, PLPP1, POFUT2, POGLUT2, POLR1C, POLR2L, POLR3C, POU3F2, PPA2, PPHLN1, PPP1R16B, PPP1R32, PPP1R9B, PPP2R2B, PPP2R3B, PRAME, PRDX3, PREB, PRELP, PRKCE, PRKCH, PRMT8, PRSS3, PSMB10, PSMC4, PSMD3, PTK6, PTPN18, PTPN2, PTPN9, PYROXD2, R3HDM2, RAB29, RAB30, RAB33B, RAB6B, RAB8B, RAD18, RAET1E, RAG2, RASGRP4, RASSF1, RBM12, RBM46, RCBTB2, RCC1L, RDX, RECQL4, REEP4, REG3A, RET, RGCC, RILP, RIMS2, RIN1, RINL, RITA1, RMND5A, RNF181, RPA3, RPL18, RPL28, RPS6KA2, RRM2, RRP15, RTN2, RUFY4, RUNDC1, SAMD13, SAMD4A, SCEL, SCGB2A2, SDCBP, SDHAF2, SEC23B, SEC63, SEMA3D, SEMA6D, SENP5, SEPTIN6, SEPTIN7, SERPINA10, SERPINE2, SFT2D2, SGCA, SGCB, SGIP1, SH3GL2, SHFL, SHOC2, SIAH2, SIGLEC7, SIRPG, SIRT6, SLC17A4, SLC22A5, SLC23A1, SLC23A3, SLC25A25, SLC25A3, SLC25A40, SLC25A41, SLC27A2, SLC27A3, SLC29A4, SLC2A8, SLC37A2, SLC39A12, SLC41A3, SLC44A5, SLC46A3, SLC51B, SLC6A5, SLCO4A1, SMAD3, SMG9, SMIM19, SMOX, SMPDL3B, SNF8, SNX5, SOCS6, SOD3, SOHLH2, SOX6, SOX8, SOX9, SPAG5, SPATA4, SPATCI, SPNS1, SPOCK1, SPRED1, SPSB1, SRF, SRM, SRPK2, SRRD, SSH3, SSRP1, ST6GALNAC6, ST7, ST8SIA3, STAT4, STIMATE, STING1, STOML2, STX1B, STX5, STXBP2, SYCE1, SYT11, SYT9, TAB2, TAMM41, TBC1D21, TBCD, TBL3, TBRG4, TBX10, TBX21, TBX22, TCF7L2, TERF2IP, TES, TEX10, TEX2, TEX45, TFAP2A, TFF2, TGFB3, THOC1, THOC3, THOP1, THRB, THRSP, THUMPD3, TIPRL, TLE1, TLE4, TMED3, TMEM107, TMEM183A, TMEM64, TMEM98, TMLHE, TMOD4, TNFSF15, TNNT2, TOMM22, TPRG1L, TPST2, TRAF3IP1, TRIM40, TRIM47, TRIP13, TRMT13, TRMT1L, TRMT2A, TRPV2, TRPV5, TSGA10, TSPAN16, TSPAN2, TSPAN32, TSPAN4, TSPAN5, TUBAL3, TUBB, TUBB3, TUBB4A, TUBGCP2, TULP4, TUT7, TXNIP, U2AF1L4, UBA6, UBASH3B, UBE2B, UBE2G1, UBE2I, UBE2O, UBQLN2, UBR2, UGDH, ULK3, UMOD, UNC13D, UQCRC2, UQCRFS1, USP1O, USP28, USP33, UVRAG, VAX2, VEGFA, VNN1, VNN3, VPS72, VRTN, VSIG8, VTI1B, VWC2, WBP11, WDFY2, WDR4, WDR60, WDTC1, WEE1, WFDC13, WFDC6, WNT2B, WRAP73, WSB1, XKR6, XPO7, XRCC5, XXYLT1, YAP1, YBX3, YY1AP1, ZBTB18, ZBTB39, ZDHHC20, ZDHHC5, ZIK1, ZKSCAN1, ZNF101, ZNF16, ZNF175, ZNF224, ZNF25, ZNF334, ZNF350, ZNF462, ZNF485, ZNF519, ZNF653, ZNF677, ZNF71, ZNF761, ZNF776, ZNF785, ZNHIT2, and ZSCAN18. In some embodiments, the immunosuppressive resistance gene is MCAM. In some embodiments, the immunosuppressive resistance gene is FOSB. In some embodiments, the immunosuppressive resistance gene is COPZ2.
[0118] In another aspect, this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 2, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. In some embodiments, increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a TGF-P- driven tumor microenvironment compared to an unmodified lymphocyte. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, Clorf35, Clorf87, C1QB, C1QL3, C1QTNF12, C3AR1, C7orf26, CACNG3, CACNG6, CALHM1, CALHM3, CAPN2, CAPZA2, CARD8, CASD1, CASK, CATSPER2, CAVIN3, CCDC106, CCDC121, CCDC153, CCDC68, CCDC70, CCDC93, CCL28, CCNL1, CCNY, CCT7, CD300C, CD300LD, CD96, CDC37, CDC42EP2, CDCA2, CDH13, CDH26, CDHR1, CDPF1, CDRT4, CEP120, CEP43, CERKL, CERS2, CES2, CFAP161, CFAP46, CGGBP1, CHID1, CHKA, CHRND, CIART, CITED2, CITED4, CLDN18, CLEC2D, CLSTN1, CLSTN3, CNDP2, COL6A2, COLCA2, COMT, COPZ2, CORO6, CPA6, CPT1C, CREB3L2, CRLF3, CRP, CSH1, CYP2C8, CYP2D6, CYP51A1, DBT, DCDC2, DCP2, DCUN1D5, DDB1, DDI2, DDX1, DDX53, DEAF1, DELEI, DEXI, DGKG, DHRS13, DKK4, DNAJB8, DNAJC11, DNTT, D0K5, DPP4, DPYSL4, DPYSL5, DSCC1, DUSP8, DYDC2, E2F2, EBP, ECU, EDN3, EGLN3, EIF1AY, EIF2AK1, ELAVL1, ELN, ELOA, ELOA2, EMC3, EMC4, ENKUR, ENOXI, EPB41L1, EPB41L4A, EPHX4, ERICH1, ESRP1, ETFDH, FAM161B, FAM207A, FAM20C, FAM76B, FAM78A, FBXL3, FBXO31, FBXO4, FCGR2A, FCN2, FCRL5, FCRLB, FGF18, FGFR1OP2, FGL1, FHL5, FIGNL1, FLU, FNDC9, FOXA3, FPGS, FREM1, FRMD8, FSD1L, FUZ, FXR1, GABBR2, GABRQ, GATAD1, GDF10, GDF5, GDF6, GGA1, GGT5, GID8, GJA10, GKAP1, GMCL1, GNA12, GNAI3, GNB5, GNL1, GNLY, GORASP1, GORASP2, GPC5, GPN2, GPR15, GPRASP2, GRIK2, GRINA, GRP, GSPT2, GTF2F1, GTPBP2, GXYLT1, H3C10, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HIC2, HINT1, HLA-DOB, HLA-DQB2, HOXB9, HOXD3, HTR1A, HYLS1, IDS, IFNA10, IFNL3, IGFBP1, IGFBP4, IGHG1, IGHV7-81, IK, IL13RA1, IL17C, IL1R2, IL2RG, IMPA1, INKA2, INO80E, INPP5J, IQCG, IQUB, ITGB7, ITLN1, ITPA, JADE1, JAKMIP1, JAML, KCNAB2, KCND1, KCNMB1, KCTD10, KCTD13, KHDRBS3, KIAA0895, KIFC2, KLC2, KLHDC1, KLHL9, KLK10, KPTN, KRT79, KRTAP10-7, KRTAP4-4, L3MBTL4, LAMB3, LCN9, LDHA, LHX2, LIAS, LIMA1, LIMD1, LMNA, LMO2, LOXHD1, LPCAT2, LRFN5, LRP11, LRP3, LRRC18, LRRC45, LRRFIP2, LRTM1, LYPD1, LYPD5, LYPD6B, MACROD1, MACROH2A1, MAFF, MAGEB6, MAGEH1, MALT1, MANF, MAP6D1, MAPK15, MAPKAPK5, MAPRE3, MARCKS, MARS1, MAST2, MAST3, MAST4, MC3R, MCAM, MCM8, MCM9, MCU, MDGA2, METTL25, MGAT4B, MICU2, MIDN, MINDY1, MIPEP, MLST8, MMD, MMP11, MMUT, MORN1, MOXD1, MPND, MPP2, MPV17L2, MPZL1, MRAP2, MRM3, MRPL3, MRPL4, MRPL41, MRPL48, MRPL51, MRPS30, MRS2, MSMP, MSX1, MSX2, MTMR1, MTPAP, MUC3A, MVB12A, MX1, MXRA8, MYBL1, MYO1A, MYOC, MY0M3, NABP2, NAF1, NAGLU, NAIF1, NAXD, NCCRP1, NCDN, NDOR1, NDUFA7, NDUFS8, NELLI, NEUROG3, NFE2L3, NFIA, NFS1, NHLRC3, NINJ2, NIPAL3, NKAP, NMD3, NONO, NPDC1, NPNT, NR5A1, NRSN2, NTN5, NUBP1, NUDT3, NUDT9, NUP54, NUP62, NUP85, NXNL2, NXPE3, NYX, OAS1, ODF3, ODF3L2, OGGI, OLIG1, OLIG3, OPCML, OR10H1, OR4A15, OR4K17, OR51E1, OR5D14, OR8B12, ORC3, OSGIN1, OSTN, OTUB2, OTUD5, OXGR1, P2RY12, PACC1, PACSIN1, PACSIN2, PAK4, PARG, PARM1, PAX9, PCDH8, PCDHA10, PCDHB13, PCDHB2, PCSK7, PCYOX1L, PDE4A, PDE9A, PDHB, PDIA3, PDZD7, PEMT, PENK, PEX16, PFDN5, PFKFB3, PFKFB4, PFKL, PGRMC1, PHACTR3, PHF7, PICALM, PIGH, PIGP, PKM, PKP1, PLA2G3, PLAT, PLCG2, PLD6, PLEKHG5, PLEKH02, PLP2, PLPPR2, PLXDC2, PMFBP1, PNMA2, PNOC, PNPT1, PGDN, POGLUT3, POLR3E, POLR3F, POU3F2, PPCDC, PPP1R12C, PPP2R1A, PPP2R2D, PPP3R2, PRDM1, PRKCD, PRLHR, PRMT2, PRNP, PRPF4, PRPF40A, PRR18, PRSS22, PSMA2, PSMD5, PTDSS1, PTGES2, PTK6, PTPN18, PTPRH, PTPRJ, PTPRS, PUM3, PWP1, PXMP4, PYROXD2, QPRT, RAB23, RAB39A, RAB42, RAB6B, RAD51B, RAFI, RAEGPS1, RASSF2, RBM24, RBM3, RBM34, RBM46, RCC1E, RCN3, RDH10, RHBDD1, RHOH, RHOXF1, RIEPE2, RIN1, RING1, RINE, RITA1, RNF126, RNF141, RNF144B, RNF220, RNF6, RO60, ROR1, RP2, RPH3AE, RPE30, RPE34, RPE6, RRS1, RSAD2, RTE8A, RUNDC1, RUNDC3A, RUSC1, RXFP3, SAFB2, SASS6, SBK1, SCG3, SCNN1A, SDF2E1, SDHAF2, SEC14E2, SEC61G, SEC63, SEE1E2, SEMA3G, SENP5, SERBP1, SERPINB5, SF1, SFRP1, SFT2D2, SFXN3, SGK1, SGO1, SH3GE2, SH3GEB1, SHH, SHKBP1, SHOC2, SIVA1, SKIE, SEC12A1, SEC1A7, SEC22A13, SEC22A7, SEC25A20, SEC25A43, SEC27A6, SEC2A13, SEC2A8, SEC37A3, SEC45A2, SEC49A4, SEF1, SMAD6, SMARCE1, SMG5, SNAP91, SNAPC2, SNRPB, SNRPG, SNX14, SNX27, SOWAHA, SPAG5, SPATS2, SPIRE1, SPRTN, SPTEC2, SRC, SRF, SSH3, SSX3, ST3GAE6, STARD7, STIM1, STK3, STK35, STMN1, STOME3, STX10, STXBP4, SUSD3, SUSD6, SYP, TAC1, TAFA5, TBC1D19, TBCK, TBE3, TBRG4, TCAF1, TCEA2, TCF19, TCP11, TCP11E1, TDG, TDP2, TEX13A, TGM4, TGOEN2, THAP11, THBD, THOC5, TIMM29, TER2, TM4SF4, TMED6, TMEM106B, TMEM178B, TMEM204, TMEM234, TMEM263, TMEM41A, TMEM86A, TMIGD2, TMPRSS1 IE, TMPRSS2, TNFAIP1, TNFAIP8L1, TNFRSF10C, TNFSF13B, TOR1B, TOX2, TPD52L2, TPM4, TPO, TPSD1, TPT1, TRAPPC10, TRAPPC8, TRIM40, TRIM55, TRIR, TRMT12, TRPC5, TRPV2, TSKS, TTC12, TTLL7, TUBA1C, TUBGCP2, TXNDC5, UBAC1, UBXN2A, UGT3A1, UIMC1, UNCI 19, UQCRFS1, USH1C, USP15, USP21, VAC14, VEGFA, VPS37B, VPS45, VRTN, WAPL, WDR1, WDR24, WDR54, WDR5B, WDR60, WDR61, WIPI2, WNT2, XAF1, YBX2, ZAP70, ZBTB5, ZC2HC1A, ZC3H3, ZCCHC8, ZDHHC1, ZDHHC13, ZFP2, ZFP36L2, ZGRF1, ZMPSTE24, ZNF175, ZNF19, ZNF205, ZNF274, ZNF428, ZNF436, ZNF502, ZNF558, ZNF624, ZNF668, ZNF71, ZNF710, ZNHIT2, and ZSWIM1.
[0119] In another aspect, this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 3, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. In some embodiments, increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a macrophage- driven tumor microenvironment compared to an unmodified lymphocyte. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABAT, ABHD12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, AL0XE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GALNT2, B3GALT4, B3GNT2, BABAM1, BAP1, BCAP31, BCCIP, BCL2L2, BCR, BDNF, BECN1, BEND2, BEND7, BLOC1S4, BMP5, BMPR1B, BRD3, BRF2, BRINP3, BTBD17, BTNL3, C10orf62, C12orf42, C14orf28, Clorf210, C2orf78, C6, C7orf31, CAB, CACNB3, CALR3, CARD14, CARD19, CASC1, CASP10, CASQ2, CBFB, CBX3, CBX4, CCDC141, CCDC148, CCDC68, CCDC8, CCDC82, CCIN, CCNG2, CCNY, CCR1, CCR10, CCR3, CD19, CD244, CD47, CD5L, CD83, CDC42EP1, CDC42EP4, CDCA2, CDCP2, CDH13, CDYL2, CEP120, CEP63, CERCAM, CERKL, CFAP100, CFAP20, CFAP410, CFAP47, CFAP91, CFHR1, CHGA, CHUK, CIRBP, CLCNKB, CLDND1, CLU, CMTR1, CNPPD1, COA3, COG1, COL22A1, COL25A1, COL6A2, COL8A2, COPB1, COPS3, COQ3, COQ8B, C0X18, CPOX, CPQ, CPT1A, CPXM1, CRACR2A, CREB3L1, CRNN, CRTAP, CRYGD, CS, CSF1, CSF1R, CSF2RB, CSF3R, CSGALNACT1, CSTF2, CTCF, CTSV, CUEDC1, CUL2, CXADR, CYB5R2, CYP20A1, CYP26A1, CYP2C19, CYP8B1, DAAM2, DAB1, DACT2, DARS1, DBN1, DCAF4L2, DCAF8, DCT, DDO, DDR1, DDX43, DDX54, DEAF1, DECR1, DENR, DESI2, DHX40, DIP2A, DKK4, DNAAF3, DNAI2, DNAJC7, DNAL1, DPP4, DPT, DPYSL4, DRG1, DSCAM, DTL, DTX2, DUS1L, DUSP12, E2F7, ECHI, EFEMP2, EFHC1, EFS, EIF2B3, EIF4A2, EIF5, ELAVL4, ELL, ELM0D3, ELN, ELOA, ELOA2, ELP3, EMC7, ENCI, ENDOV, ENPP3, ENTPD5, EOLA2, EPB41L1, EPHB6, EPN1, ERCC6, ESRP1, ESRRA, ETFDH, EX01, EXOC3, EXOSCIO, EXOSC8, EYS, F3, FABP6, FAM117A, FAM117B, FAM118B, FAM161B, FAM200A, FAM20A, FAM47A, FARSA, FBLN1, FBLN5, FBP1, FBXO24, FBXO40, FCHO1, FCRL5, FCRLB, FECH, FEM1B, FGF3, FGG, FKBP6, FLVCR1, FMNL1, FM05, FNIP1, FOS, FOSB, FOXD4, FOXJ1, F0XM1, FOXN3, FOXRED2, FREM1, FRMD5, FRS2, FSTL1, FSTL4, FSTL5, FTMT, FXR2, G6PD, GABPB1, GABRB1, GALNT7, GANAB, GAPDHS, GATA2, GBP4, GCAT, GCGR, GCNT7, GCSAML, GDF2, GDF6, GET4, GGA1, GHDC, GINS4, GK, GKAP1, GLB1, GLRA2, GLRX5, GLYR1, GMCL2, GNA14, GNAT2, GNB3, GNL1, GNL3, GPAT4, GPC3, GPC4, GPC5, GPR84, GRHL3, GRHPR, GRIA4, GRID1, GRIK3, GRK4, GRM3, GRM8, GRN, GSDME, GSK3A, GSN, GTF2A1, GTF2B, GTF2E1, GUCY1B1, GYSI, HABP2, HADHA, HADHB, HDAC10, HEPHL1, HERPUD2, HES1, HEXD, HHIPL2, HINT2, HLA-C, HOMER3, HOOK3, HOXB6, HOXD3, HPS3, HPS5, HSD11B2, HSD17B13, HSD17B7, HSD3B1, HSPB9, HTR2B, IFNA6, IFNL1, IFT27, IGHA1, IGSF1O, IGSF21, IL1ORB, IL12RB2, IL13RA1, IL21, IL26, IL7R, ILVBL, IMPG1, INA, INHA, INSL4, INTU, IQUB, IRAG1, IRAKI, IREB2, IRF3, IRF5, IRX3, ITFG1, ITGB2, ITGB7, ITIH5, ITM2B, JAKMIP1, KANSL3, KAT5, KCNG3, KCNJ14, KCNK12, KCNK2, KCNK9, KCNMB3, KCTD12, KCTD4, KIAA2013, KIF2C, KIF3A, KIF3B, KIFC2, KITLG, KLC3, KLHDC2, KLHL13, KLHL21, KLHL8, KLK1, KRT6A, KXD1, L3MBTL4, LAD1, LAMP1, LAP3, LARS2, LDLRAD3, LGMN, LHX2, LHX4, LIMA1, LIMD1, LIPH, LIPT1, LKAAEAR1, LNPEP, LOXHD1, LOXL3, LTA4H, LTBR, LUC7L2, LYL1, LZTS2, MAG, MAN1A1, MANEA, MANF, MAP3K7, MAP4K5, MAP6D1, MAPK15, MAPK8, MAPKAPK5, MAPKBP1, MARS2, MATN2, MBLAC1, MCAM, MCOLN2, MCOLN3, MDH1B, MDM4, MEAK7, MECP2, MED1, MEOX1, METAP1, MFAP3L, MFNG, MITD1, MLEC, MLH1, MLLT3, MMP10, MMP16, MMS19, MNAT1, MON1B, MPP2, MPP7, MRM3, MRPL19, MRPL3, MRPL47, MSLN, MSRA, MTARC2, MTHFR, MTMR4, MTMR6, MUTYH, MVP, MYBL1, MYCN, MYO1A, MYO5C, MYOC, MYOM3, MYORG, MYT1, NAGLU, NAMPT, NCKIPSD, NDC80, NDRG4, NDUFAF7, NDUFB6, NDUFS8, NDUFV2, NECTIN4, NELFA, NFIB, NIF3L1, NIPAL1, NIPBL, NMD3, NOC2L, NPLOC4, NPY, NPY2R, NQO1, NR2E1, NR6A1, NT5DC2, NTM, NTN5, NTNG1, NUDT8, NUP210, NXPE3, OAS1, OAS2, ODF2, OGA, OGFRL1, OLFM2, OLFML1, OLFML2B, OLIG1, OLR1, OMP, OR4D1, OR4S2, OR52N5, OR52W1, OR5B3, OR9Q1, ORMDL2, OSBPLIO, OSGEPL1, OTX1, OXSR1, P2RX6, P2RY1, PACS2, PAEP, PAFAH1B2, PAK4, PARL, PARP9, PARS2, PC, PCDHA1, PCDHA6, PCDHGA2, PCDHGA5, PCSK2, PDCD6, PDE4A, PDIA3, PDK2, PDZD9, PENK, PFKL, PGAM5, PGK1, PGM2, PHF11, PHYH, PICALM, PIK3R1, PIP5K1B, PLAGL1, PLCD4, PLD1, PLEK, PLEKHG5, PLEKHS1, PLIN1, PLK1, PLPPR2, PM20D1, PNMA8A, POLE, POLI, POLR2K, POLR3F, POU3F2, PPA2, PPIG, PPM1D, PPP1R12C, PPP1R16B, PPP1R32, PPP2R3C, PPP2R5C, PPP2R5D, PRAC1, PRAM1, PRAME, PRDM1, PRDX3, PRKAA2, PRKAG2, PRKAR1B, PRKCE, PRMT1, PROC, PRRT2, PRSS45P, PRSS48, PSAPL1, PSD3, PSEN1, PSMC4, PSMD14, PTBP3, PTCD2, PTDSS1, PTGER2, PTPN12, PTPRN, PUF60, PUM1, PYDC1, PYGB, RAB1A, RAB33B, RAB42, RABL6, RAD18, RAD51, RAET1G, RALBP1, RASSF4, RBBP7, RBM12, RBM38, RBM4, RBM46, RBM4B, RBX1, RCBTB2, RCC1L, RDH10, RDH12, REC8, REN, RET, RFC2, RFC5, RFX4, RGS16, RHAG, RHEX, RHOBTB2, RICTOR, RIMBP2, RIMS3, RIOK3, RIPK1, RIPK2, RIT1, RMDN3, RNASEH2B, RNF114, RNF148, RNF213, RNF6, RNPEPL1, RO60, RORA, RPL30, RPS14, RPS4X, RPS6KA2, RPS6KB1, RSRC1, RTF1, RTN1, RUBCN, RUNDC1, RUNX3, S1OOPBP, SAMHD1, SCAMP2, SCIN, SCNN1D, SCRN2, SDSL, SEC63, SEL1L3, SELL, SENP3, SEPTIN10, SEPTIN8, SERINC3, SERPINF1, SGK3, SGMS1, SH3GLB1, SHOC2, SIAH2, SIGLEC10, SIGLEC12, SIGLEC7, SIRPG, SIRT5, SKAP1, SKIL, SLC15A3, SLC16A1, SLC16A7, SLC18A2, SLC1A7, SLC20A2, SLC22A2, SLC22A23, SLC22A24, SLC23A2, SLC25A19, SLC25A25, SLC25A47, SLC26A2, SLC2A4, SLC2A8, SLC36A3, SLC37A2, SLC37A3, SLC39A14, SLC43A1, SLC5A11, SLC5A12, SLC5A7, SLC6A7, SLC7A1, SLCO1A2, SLITRK3, SMARCD3, SMOX, SNCAIP, SNTA1, SOCS6, SOX9, SPATA2, SPCS3, SPN, SPNS2, SPOCK3, SPOUT1, SPRR4, SRC, SRD5A3, SRFBP1, SRMS, SSMEM1, SSX2IP, ST7L, STEAP1, STIM1, STK11, STK17A, STK17B, STK3, STK39, STXBP1, STXBP2, STXBP3, STXBP5, SUGCT, SUN5, SUSD2, SYNCRIP, TAB2, TBC1D10A, TBC1D22B, TBC1D9B, TBCD, TBL1X, TBX6, TON, TCF25, TCTN2, TDGF1, TEAD2, TESK1, TESK2, TEX48, TFAP2A, TFAP4, TGFB1, TGFBI, TGOLN2, THAP3, THAP7, THEM4, THOC1, THOC7, THOP1, THSD4, TIAM2, TICAM2, TLE6, TLK1, TM9SF4, TMED2, TMEM259, TMEM62, TMIE, TMLHE, TMOD1, TMTC3, TNF, TNS1, TOMM70, TRAF3IP1, TRAM1, TRAP1, TRAPPC3, TRAPPC4, TRAPPC8, TRAPPC9, TRIB3, TRIM32, TRMT13, TRPM3, TRPV4, TSGA10, TSPEAR, TSPYL6, TTC13, TTC16, TTC26, TTC38, TTC8, TTF2, TTLL2, TTLL7, TUBA3E, TUBA8, TUBB3, TUBB4B, TUFM, TYMS, UBE2O, UBE2Z, UBE3A, UBE3C, UBOX5, UGDH, UGP2, UGT3A1, UGT8, UMOD, USP1, USP49, UXS1, VANGL2, VASN, VEGFA, VIL1, VPS45, VWA1, WAPL, WDR37, WDR63, WDR90, WDR91, WNT4, WWOX, XRCC5, YBX2, YME1L1, ZBTB14, ZBTB18, ZBTB46, ZC3H10, ZC3H11A, ZCCHC8, ZDHHC11, ZDHHC15, ZDHHC6, ZFYVE21, ZGRF1, ZMAT3, ZNF165, ZNF18, ZNF19, ZNF20, ZNF224, ZNF25, ZNF277, ZNF334, ZNF350, ZNF354C, ZNF396, ZNF398, ZNF436, ZNF461, ZNF467, ZNF496, ZNF560, ZNF565, ZNF566, ZNF597, ZNF610, ZNF623, ZNF668, ZNF74, ZP1, ZPLD1, ZSCAN25, and ZSWIM2.
[0120] In another aspect, this disclosure provides a modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 4, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. In some embodiments, increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a regulatory T cell-driven tumor microenvironment compared to an unmodified lymphocyte. In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, ADAT1, ADGRE5, ADIPOR2, AD0RA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, A0C1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1, B9D2, BAB AMI, BAG5, BCHE, BLK, BMPR1B, BPIFC, BRF2, BSND, BTNL3, BYSL, C10orf82, C18orf25, C18orf54, Clorfll5, Clorf43, Clorf56, C1QTNF2, C1R, C2CD2, C5orfl5, C6orfl20, CAB, CA5B, CA8, CA9, CABS1, CALCOCO1, CALCR, CALHM3, CAMK2A, CAMLG, CARD8, CASP1, CASP7, CASTOR1, CBX7, CCDC110, CCDC69, CCDC82, CCDC84, CCL2, CCL21, CCR8, CCSER1, CD151, CD300LF, CD48, CD83, CD86, CDH7, CDK1, CDPF1, CDR2, CELF1, CEP63, CERS1, CES3, CFAP410, CGGBP1, CHAF1B, CHMP2A, CHMP7, CHRM1, CHST9, CISH, CLC, CLDN6, CLEC5A, CLIC5, CLP1, CLTRN, CLUAP1, CMTM7, CNTF, COG3, COL25A1, COL8A2, C0MMD4, COPZ1, COPZ2, COQ4, COX6B2, CPLX2, CRACR2B, CROT, CRTAC1, CRY2, CSF1, CSNK1G2, CST9, CSTF3, CTDP1, CTNNA2, CTSF, CXCR6, CXXC1, CYP27A1, CYP2D6, CYTL1, DAXX, DBN1, DDHD2, DDX24, DES, DEXI, DGLUCY, DHX36, DNAI2, DNAJA2, DNAJB5, DNAJB6, DNAJC11, DNAJC27, DNAJC6, D0K5, D0K6, DRGX, DUS1L, DUSP5, DZIP1L, ECI2, EFCAB7, EGFL6, EGLN3, EIF3K, EIF4EBP1, ELSPBP1, EMC3, EPDR1, EPN1, EPO, ERFE, ERVK3-1, ESMI, F2RL2, FAAP100, FADS1, FAM172A, FAM53C, FAM71C, FAM81A, FBLN5, FBXL16, FBXO7, FBXW11, FCGR3A, FCRL5, FDFT1, FETUB, FEZF2, FGF10, FGF19, FGL1, FKBP5, FKBP9, FMOD, FNDC9, FOSB, FRMD3, FRMD5, FRZB, FUT3, GAB3, GABRA4, GABRG2, GALNT2, GALNTL6, GAS7, GBA2, GBP6, GEMIN8, GET1, GFM2, GFRA3, GK2, GLIPR1, GLRA2, GLRB, GLYCTK, GNA14, GNB3, GNL3, G0LM2, GPATCH2L, GPATCH3, GPM6B, GPR176, GPR45, GRAMD1B, GRB7, GRK2, GRK7, GSTM3, GXYLT1, GZMM, HAPLN3, HAUS2, HBG2, HCRTR1, HDAC8, HDGF, HEMK1, HERPUD1, HESX1, HEXA, HMHB1, H0XB5, H0XB6, H0XD3, H0XD4, HPGDS, HSD17B6, HSD17B8, HSPA2, HTN1, HTR5A, IFITM3, IFNA10, IGF1, IGFALS, IGHM, IL11RA, IL17A, IL17RE, IL2RB, IL4, INSL6, ISL2, ISM2, IST1, ITPRID2, JAML, JUN, JUNB, KBTBD7, KCNA6, KCNN3, KEAP1, KIF3A, KIFC2, KIR2DL1, KLHDC2, KLHDC7B, KLHL9, KRT19, KRT6A, LARS2, LCK, LCN1, LDAH, LDLRAD4, LETMD1, LHX4, LHX9, LIN28A, LIPG, LNX1, LPAR5, LPL, LRFN3, LRRC15, LRRC2, LRRC34, LRRC42, LTBR, LYZ, MAF1, MAGOH, MAN1A1, MAN1B1, MAP2K6, MAP3K7, MAP4K5, MAPKAPK2, MAPKAPK5, MARCHF1, MARCHF2, MBNL1, MBP, MC3R, MCAM, MED26, MEIS3, METTL27, METTL2B, MIA2, MIF, MIPOL1, MLKL, MMD, MMP10, MOB4, MPHOSPH8, MPND, MPZL1, MR1, MRAS, MRM3, MRPL21, MRPS24, MRPS28, MS4A3, MS4A5, MSRA, MTA2, MTA3, MTHFD2, MTMR3, MTPAP, MTRF1L, MUS81, MYCL, MYCN, MYL10, MYL9, MYOC, MYOM3, MYORG, NAA80, NAPSA, NARF, NAT1, NCK1, NDE1, NDOR1, NDUFA13, NDUFA4, NEK5, NELFE, NFIB, NFKBIB, NINJ2, NIPAL1, NKAIN2, NKAP, NMB, NOC4L, NPIPB15, NRARP, NRSN2, NTF3, NXNL2, OBP2A, OLR1, OR10AG1, OR10K2, OR14C36, OR1F1, OR2M3, OR2T8, OR5C1, OR7A5, OR9Q1, ORAI3, ORC2, ORM1, ORM2, OSGIN1, OSR2, OTUD5, P3H4, P4HA3, P4HTM, PAK2, PAK4, PBX2, PCDHA2, PCDHB12, PCDHGA2, PDYN, PFDN4, PFKP, PFN4, PGK1, PGLYRP1, PHF23, PHF7, PHKG1, PHOSPHO1, PI15, PI3, PI4KB, PITHD1, PKIA, PKNOX2, PLA2G3, PLA2G7, PLAUR, PLEKHA8, PLEKHO2, PLET1, PLOD2, PNPT1, POPDC3, PPM1D, PPME1, PPP1R2, PPP1R32, PPP2R2B, PPP2R3C, PPP2R5C, PPP6R2, PRKCB, PRKRIP1, PRKY, PRMT8, PRSS3, PRUNE2, PSCA, PSG1, PTGER3, PTK6, PTOV1, PTPRO, PTTG1IP, PUDP, PWWP3B, PYCARD, PYGB, QPCT, QPRT, RABI IB, RAB25, RAB28, RAB34, RAB40B, RABEP2, RADU, RAET1E, RAMP1, RARS1, RAX2, RBBP5, RCHY1, RCN3, REEP2, RFC4, RFPL2, RFX3, RIBCI, RINL, RIPK4, RLBP1, RNASE9, RNASET2, RNF111, RNF112, RNF144B, RNF24, RNF38, RNF7, ROPN1L, RP9, RPL6, RPS2, RPS3A, RPS6, RRAGA, RRAGD, RRP1, RRP9, RSAD1, RUBCN, RUNX1T1, RUVBL1, SAMHD1, SARS1, SCNN1A, SCNN1B, SCNN1G, SEL1L2, SELENBP1, SEPHS1, SEPTIN10, SEPTIN12, SERINC2, SERPINA3, SERPIND1, SERPINE1, SERPINE3, SETD3, SFRP4, SGK2, SH3KBP1, SHARPIN, SHISA3, SHOC2, SHOX, SIGLEC7, SIRPB2, SIRPG, SIRT3, SKIL, SLC13A1, SLC14A1, SLC20A2, SLC22A13, SLC22A31, SLC22A8, SLC25A1, SLC25A46, SLC25A47, SLC25A48, SLC2A8, SLC37A3, SLC39A7, SLC5A12, SLC6A19, SLC6A7, SLC7A9, SLCO1A2, SLFNL1, SMG9, SMPX, SNORC, SNRNP25, SNRPB, SNRPN, SNX16, SOAT1, SOCS5, SPATA22, SPATS2, SPC25, SPG21, SPINT1, SPINT2, SPNS2, SPP2, SQOR, SRC, SRFBP1, SRP54, SRP9, SRSF9, SSBP2, SSPN, STAP1, STARD7, STIM1, STK11, STX8, SULT4A1, SUMF2, SURF6, SYMPK, SYNCRIP, SZT2, TAS2R40, TAS2R60, TBCC, TBRG4, TBX20, TBX3, TC2N, TCEA1, TCEA2, TCF19, TCF7L2, TCN2, TCTN1, TDP2, TENT5C, TEX35, TFCP2L1, TFDP2, TGFB3, THAP12, TIAM2, TICAM2, TIPIN, TKT, TLE4, TM2D2, TM9SF3, TMEM106B, TMEM143, TMEM160, TMEM211, TMEM237, TMEM270, TMEM30A, TMEM39B, TMEM45A, TMEM68, TMPRSS3, TNFSF12, TOMM70, TOR1AIP1, TOX, TPP1, TPRKB, TPST2, TRAPPC12, TRDMT1, TRIM10, TRIM47, TRIM63, TRIP10, TRMO, TRMT44, TRPV4, TSN, TSPAN31, TSPEAR, TSSK3, TTBK2, TTC32, TUBA3C, TUBA3D, TUT7, TYW3, UBA1, UBASH3B, UBE2S, UBXN2A, UCHL3, UCHL5, UQCR10, USP1, USP19, VAT1L, VMA21, VPS29, VPS36, VSTM2A, VWA1, WAS, WDFY1, WDR4, WDR59, WDR63, WDR78, WNT11, WNT3A, WNT9A, YAF2, YJU2, ZBTB48, ZC3HAV1L, ZCCHC2, ZCCHC7, ZFP36L2, ZIC3, ZNF165, ZNF830, ZP2, ZSCAN21, ZSCAN9.
[0121] In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, HOXB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPL0C4, NPNT, NRSN2, NTN5, NXNL2, 0AS1, 0LFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
[0122] In some embodiments, the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MYO1A, MYOC, MYOM3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
B. Chimeric Antigen Receptors and T Cell Receptors
[0123] Provided herein, in some embodiments, are modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a chimeric antigen receptor (CAR). In some embodiments, the therapeutic protein comprises a T cell receptor (TCR).
Chimeric Antigen Receptors (CARs)
[0124] In some embodiments the term “chimeric antigen receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as an intracellular signaling domain) comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the stimulatory molecule is TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4- IBB, or CD3-zeta. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the stimulatory molecule is 4-IBB. In some embodiments, the stimulatory molecule is CD28. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below (also referred to as a “co stimulatory signaling domain”). In some embodiments, the costimulatory molecule is chosen from a costimulatory molecule described herein, e.g., 0X40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD1 la/CD18), ICOS and 4-IBB (CD 137), or any combination thereof. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule (a primary signaling domain). In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule (a costimulatory signaling domain) and a functional signaling domain derived from a stimulatory molecule (a primary signaling domain).
[0125] In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the scFv domain during cellular processing and localization of the CAR to the cellular membrane.
[0126] In some embodiments, the extracellular domain of the CAR binds to a tumor antigen. In some embodiments, the tumor antigen is HER2. In some embodiments, the tumor antigen is Claudin-6 (CLDN6). In some embodiments, the tumor antigen is expressed at a low level in a cancer cell or tumor cell. In some embodiments, the cancer cell or the tumor cell is an antigen-low cancer cell or an antigen-low tumor cell.
[0127] In some embodiments, the CAR binds to CLDN6. In some embodiments, the CAR is a CLDN6-CAR. The CLDN6-CAR can comprise any one of the nucleotide and/or amino acid sequences described in US 2022/0306711 Al, US 2022/0339193 Al, US 10,561,686 B2, and Mackensen et al., Nat. Med., 2023, 29:2844-2853, all of which are herein incorporated by reference in their entireties.
[0128] In some embodiments, the modified lymphocyte comprises a nucleic acid sequence encoding a CAR and further comprises a nucleic acid sequence encoding an immunosuppressive resistance gene described herein.
T Cell Receptors (TCRs)
[0129] In some embodiments, the modified lymphocyte expresses a TCR. In some embodiments, the TCR is a disulfide-linked membrane- anchored heterodimer present on T cell lymphocytes, and the majority of T cells are aP T cells having a TCR consisting of an alpha (a) chain and a beta (P) chain. In some embodiments, each chain comprises a variable (V) and a constant (C) domain, wherein the variable domain recognizes an antigen, or an MHC -presented peptide. TCRa and TCRP chains with a known specificity or affinity for specific antigens, e.g., tumor antigens described herein, can be introduced to a T cell using the methods described herein. TCRa and TCRP chains having increased specificity or affinity for a particular antigen can be isolated using standard molecular cloning techniques known in the art. In some embodiments, other modifications that increase specificity, avidity, or function of the TCRs or the engineered T cells expressing the TCRs can be readily envisioned by the ordinarily skilled artisan, e.g., promoter selection for regulated expression, mutations in the antigen binding regions of the TCRa and TCRP chains. Any isolated or modified TCRa and TCRP chain can be operably linked to or can associate with one or more intracellular signaling domains described herein. In some embodiments, signaling can be mediated through interaction between the antigen-bound ab heterodimer to CD3 chain molecules, e.g., CD3zeta (z).
[0130] In some embodiments, a smaller subset of T cells expresses a TCR having a (y) gamma chain and a delta (6) chain. In some embodiments, gamma-delta (y 6) T cells make up 3-10% of circulating lymphocytes in humans, and the Vd2+ subset can account for up to 95% of y6 T cells in blood. In some embodiments, V62+ cells recognize non-peptide epitopes and do not require antigen presentation by major histocompatibility complexes (“MHC”) or human leukocyte antigen (“HLA”). In some embodiments, the majority of V62+ T cells also express a Vy9 chain and are stimulated by exposure to 5-carbon pyrophosphate compounds that are intermediates in mevalonate and non-mevalonate sterol/isoprenoid synthesis pathways. In some embodiments, the response to isopentenyl pyrophosphate (5-carbon) is universal among healthy human beings. In some embodiments, another subset of y6 T cells, V61+, make up a much smaller percentage of the T cells circulating in the blood, but are commonly found in the epithelial mucosa and the skin. In some embodiments, y6 T cells have several functions, including killing tumor cells and pathogen-infected cells. In some embodiments, stimulation through the y6 TCR improves the capacity for cellular cytotoxicity, cytokine secretion and other effector functions. In some embodiments, the TCRs of y6 T cells have unique specificities and the cells themselves occur in high clonal frequencies, thus allowing rapid innate-like responses to tumors and pathogens. See, e.g., Park and Lee, Exp Mol Med. 2021 Mar;53(3):318-327., which is incorporated herein by reference.
[0131] In some embodiments, the modified lymphocyte comprises a nucleic acid sequence encoding a TCR and further comprises a nucleic acid sequence encoding an immunosuppressive resistance gene described herein.
C. Exogenous Nucleic Acids and Vectors
Expression Cassettes
[0132] Provided herein, in some embodiments, are modified lymphocytes comprising exogenous nucleic acids encoding an immunosuppressive resistance gene described herein. In some embodiments, the modified lymphocyte comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the modified lymphocyte comprises an expression cassette comprising nucleic acids encoding one or more immunosuppressive resistance genes.
[0133] In some embodiment, the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in the same vector. In some embodiments, the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in the same expression cassette. In some embodiments, the immunosuppressive resistance gene and the nucleic acid encoding the therapeutic protein are in different vectors.
[0134] In some embodiments, the immunosuppressive resistance gene comprises any of the genes identified in Tables 1-6. As used herein, where reference to a specific gene of Tables 1- 6 is mentioned, it is intended that the use of the coding sequence for the full-length protein, a fragment having a deletion or truncation, or a variant having one or more substitutions in the amino acid, is intended. In some embodiments, the nucleic acid encodes a full-length protein. In some embodiments, the nucleic acid encodes a functional fragment of a truncated protein. In some embodiments, the functional fragment of the truncated protein comprises one or more functional domains of the protein. In some embodiments, the functional fragment of the truncated protein may comprise catalytic activity. In some embodiments, the functional fragment of the truncated protein may facilitate protein-protein, protein-DNA, or protein- RNA interactions. In some embodiments, the immunosuppressive resistance gene encodes a variant protein having one or more substitutions in the amino acid. In some embodiments, the nucleic acid encodes a protein sequence having a deletion or truncation in the N terminus. In some embodiments, the nucleic acid encodes a protein sequence having a deletion or truncation in the C terminus. In some embodiments, the nucleic acid encodes a protein having of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, or at least 125 amino acids.
[0135] In some embodiments, the expression cassette includes more than one effectorenhancing gene, or functional fragment or variant thereof. In some embodiments, the expression cassette encodes at least two immunosuppressive resistance genes. In some embodiments, the expression cassette encodes one, two, three, four, or five immunosuppressive resistance genes. In some embodiments, the expression cassette encodes between about one and about five immunosuppression resistance genes. [0136] In some embodiments, the modified lymphocyte comprises two or more exogenous nucleic acids encoding two or more of the immunosuppressive resistance genes described herein. In some embodiments, each immunosuppressive resistance gene is encoded in a separate expression cassette. In some embodiments, the modified lymphocyte comprises a first expression cassette encoding a first immunosuppression resistance gene, and a second expression cassette encoding a second immunosuppressive resistance gene. In some embodiments, the modified lymphocyte comprises two, three four, or five expression cassettes, wherein each expression cassette encodes a unique immunosuppression resistance gene.
[0137] In some embodiments, the expression cassette comprises a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene. In some embodiments, the promoter is heterologous. In some embodiments the promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate-early enhancer and chicken betaactin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
[0138] In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte. In some embodiments, the expression cassette comprising the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus. In some embodiments, the safe harbor locus is selected from the group consisting of AAVS1, hROSA26, and CCR5. In some embodiments, the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
[0139] In some embodiments, the expression cassette further comprises a therapeutic protein. In some embodiments, the therapeutic protein comprises a CAR or a TCR.
[0140] Also provided herein are vectors comprising the nucleic acid encoding an immunosuppressive resistance gene set forth in any one of Tables 1-6. In some embodiments, the nucleic acid encodes a full-length protein or a functional fragment or variant thereof. In some embodiments, the vector comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene. In some embodiments the vector further comprises a nucleic acid encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a CAR or a TCR. In some embodiments, the vector comprises an expression cassette comprising the nucleic acid encoding the CAR or TCR. In some embodiments the vector comprises an expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR. In some embodiments, the vector comprises a first expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene and a second expression cassette comprising the nucleic acid encoding the CAR or TCR.
[0141] In some embodiments, the vector further comprises nucleic acid encoding a drugresistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene. In some embodiments, the drug-resistance gene is selected from the group consisting of neomycin resistance gene (NeoR), kanamycin resistance gene (NPTII), puromycin-N-acetyl Transferase (PAC), aminoglycoside phosphotransferase (APH), and blasticidin S deaminase (BSD). In some embodiments, the drug-resistance gene is a puromycin-N-acetyltransferase (PAC) gene. In some embodiments, the surface expressed safety switch gene is selected from the group consisting of CD20, HER2, EGFR, full length NGFR, and truncated NGFR.
[0142] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus. In some embodiments, the vector is an episomal or non-integrating vector. In some embodiments, the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
[0143] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is delivered to a lymphocyte by electroporation or cell squeezing. In some embodiments, the non-viral vector is encapsulated in nanoparticles or liposomes.
D. Lymphocytes
[0144] Provided herein, in some embodiments, are modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein. [0145] In some embodiments, the therapeutic protein is a CAR or a TCR. In some embodiments, the CAR or the TCR binds to a tumor antigen. In some embodiments, the tumor antigen is HER2 or Claudin-6. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, FOSB, GSDME, IL26, LIMA1, LTBR, PTK6, SKIL, SRC, STK11, and YBX2. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and the immunosuppressive resistance gene ZBTB46. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and the immunosuppressive resistance gene LTBR. In some embodiments, the modified lymphocyte comprises a CAR that binds to HER2, and an immunosuppressive resistance gene selected from the group consisting of LTBR, cJUN, mbIL15, and TGFBR2dn. In some embodiments, the modified lymphocyte comprises a CAR that binds to CLDN6 and the immunosuppressive resistance gene LTBR.
[0146] In some embodiments, the modified lymphocyte comprises a mutant allele of the immunosuppressive resistance gene LTBR. In some embodiments, the mutant allele encodes for a non-functional LTBR. In some embodiments, the non-functional LTBR is LTBR-del. LTBR-del is a non-functional, truncated version of LTBR lacking the intracellular signaling domain. In some embodiments, lack of an intracellular signaling domain results in the inability of LTBR to transmit signaling even when the extracellular part binds a ligand. [0147] In some embodiments, the modified lymphocyte is a T cell, a NK cell, or a NK T cell. In some embodiments, the modified lymphocyte is a CD3+ lymphocyte. In some embodiments, the modified lymphocyte is a TCRaP+ CD4" CD8" T cell. In some embodiments, the modified lymphocyte is a CD4+ T cell or a CD8+ T cell. In some embodiments, the modified lymphocyte is a CD4+ T cell. In some embodiments, the modified lymphocyte is a CD8+ T cell. In some embodiments, the CD4+ T cell or a CD8+ T cell expresses an aP TCR. In some embodiments, the modified lymphocyte is a y5 T cell. In some embodiments, the y5 T cell expresses a y5 TCR. In some embodiments, the modified lymphocyte is a human lymphocyte. [0148] In some embodiments, the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell. In some embodiments, the modified lymphocyte is a regulatory T cell.
[0149] In some embodiments, the modified lymphocyte is derived from a cell in culture. In some embodiments, the modified lymphocyte is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is engineered to knock out or silence the expression of TCR and HLA proteins. In some embodiments, the iPSC is further engineered to express immune receptors, cytokines, chemokines, or other immune regulatory factors to enhance anti-tumor function.
[0150] In some embodiments, the modified lymphocyte is derived from a lymphocyte isolated from an individual. In some embodiments, the modified lymphocyte is derived from a lymphocyte isolated from a blood sample obtained from an individual. In some embodiments, the modified lymphocyte is derived from a lymphocyte isolated from a tumor sample obtained from an individual. In some embodiments, the modified lymphocyte is derived from a tumor infiltrating lymphocyte (TIL). In some embodiments, the modified lymphocyte is a tumor infiltrating lymphocyte (TIL). In some embodiments, the modified lymphocyte is a cell therapy.
E. Compositions Comprising Modified Lymphocytes
[0151] In one aspect, provided herein is a pharmaceutical composition comprising modified lymphocytes that are engineered to express an increased level of an immunosuppressive resistance gene set forth in any one of Tables 1-6. In some embodiments, cells in the composition have anti-tumor activity. In some embodiments, the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. In some embodiments, the pharmaceutical composition comprises a modified lymphocyte comprising nucleic acid encoding an immunosuppressive resistance gene and further comprises a nucleic acid encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a CAR or TCR. In some embodiments, the pharmaceutical composition comprises a modified T cell, a modified NK cell, or a modified NK T cell. In some embodiments, the pharmaceutical composition comprises a modified CD3+ lymphocyte. In some embodiments, the pharmaceutical composition comprises a modified TCRaP+ CD4- CD8- T cell. In some embodiments, the pharmaceutical composition comprises a modified CD4+ T cell or a modified CD8+ T cell. In some embodiments, the pharmaceutical composition comprises a modified y5 T cell. In some embodiments, the pharmaceutical composition comprises a modified human lymphocyte.
[0152] In some embodiments, the composition is in the form of a liquid. In some embodiments, the liquid is a cellular suspension. In some embodiments, the liquid is useful for delivery by injection. In some embodiments, the liquid comprises a cell culture media. In some embodiments, a composition for administration by injection, in addition to the modified lymphocytes, contains one or more excipients selected from the group consisting of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent.
IL METHODS OF INCREASING LYMPHOCYTE PROLIFERATION IN IMMUNOSUPPRESSIVE CELLULAR ENVIRONMENTS
[0153] In some embodiments, provided herein are methods of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 1-6. In some embodiments, the immunosuppressive cellular environment comprises a tumor microenvironment (TME). In some embodiments, the immunosuppressive cellular environment recapitulates the in vivo immunosuppressive cellular environment within the TME. In some embodiments, the immunosuppressive cellular environment is an in vitro immunosuppressive cellular environment. In some embodiments, the immunosuppressive cellular environment established in and around tumors comprises adenosine driven immunosuppression, TGF-P driven immunosuppression, regulatory T cell (Treg) driven immunosuppression, and/or macrophage driven immunosuppression.
[0154] In some embodiments, the method comprises modifying a population of human lymphocytes with a lentiviral vector comprising an expression cassette comprising nucleic acids encoding an immunosuppressive resistance gene set forth in Tables 1-6, and optionally, a CAR or TCR. In some embodiments, the modified lymphocytes described herein are expanded. In some embodiments, the modified lymphocytes are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days).
[0155] In some embodiments, the method comprises increasing lymphocyte proliferation in an adenosine driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene conferring increased lymphocyte proliferation in an adenosine driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 1.
[0156] In some embodiments, the method comprises increasing lymphocyte proliferation in a TGF-P driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a TGF- P driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 2.
[0157] In some embodiments, the method comprises increasing lymphocyte proliferation in a Treg driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a Treg driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 3.
[0158] In some embodiments, the method comprises increasing lymphocyte proliferation in a macrophage driven immunosuppressive cellular environment. In some embodiments, the immunosuppressive resistance gene conferring increased lymphocyte proliferation in a macrophage driven immunosuppressive cellular environment is selected from an immunosuppressive resistance gene set forth in Table 4.
[0159] In some embodiments, the method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene described herein comprises the steps of: (i) collecting peripheral blood mononuclear cells (PBMCs) from an individual, (ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cell from the PBMCs of step (i), (iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and (iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene. In some embodiments, the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR). In some embodiments, the exogenous nucleic acid further comprises a T cell receptor (TCR). In some embodiments, the peripheral blood mononuclear cells are obtained from leukapheresis. In some embodiments, the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection. In some embodiments, the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element. In some embodiments, the transduced lymphocytes are enriched by positive selection. In some embodiments, the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin. [0160] In some embodiments, the CD8+ and CD4+ T cells are isolated sequentially. In some embodiments, the naive CD4+ T cells are differentiated into activated CD4+ T cells. In some embodiments, the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell). In some embodiments, the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
III. SCREENING METHODS
[0161] In one aspect provided herein are methods of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising: (i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual, (ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes, (iii) transiently stimulating the transduced lymphocytes, (iv) exposing the transduced lymphocytes to an immunosuppressive environment, (iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and (v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene. In some embodiments, the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
[0162] In some embodiments, the method comprises a genome-scale gain-of-function screen in primary human CD4+ and CD8+ T-cells.
[0163] In some embodiments, the lymphocyte population comprising a mixture of CD4+ and CD8+ cells is obtained from a human blood sample. In some embodiments, the mixture of CD4+ and CD8+ cells is obtained from an enriched apheresis product. In some embodiments, the enriched apheresis product comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the human blood sample is obtained from a healthy blood donor. In some embodiments, the CD8+ and CD4+ T cells are isolated from the PBMCs sequentially. In some embodiments, the PBMCs are not pooled together prior to the isolation of the CD8+ and CD4+ T cells. In some embodiments, the CD8+ and CD4+ T cells are isolated from the PBMCs sequentially. In some embodiments, isolating the CD8+ and CD4+ T cells comprises a first step of isolating CD8+ T cells from the PBMCs using magnetic selection of the CD8 protein, followed by a second step of isolating the CD4+ T cells from the PBMCs using magnetic selection of the CD4 protein. In some embodiments, the method further comprises isolating monocytes from the PBMCs following the isolation of CD8+ and CD4+ T cells from the PBMCs. In some embodiments, the method further comprises isolating naive CD4+ T cells from the PBMCs using magnetic selection of the CD3 protein, the CD4 protein, and the CD45RA protein. In some embodiments, the isolated mixture of CD4+ and CD8+ cells are cultured in T cell media supplemented with recombinant human IL-2.
[0164] In some embodiments, the CD4+ and CD8+ T cells are activated by culturing the CD4+ and CD8+ T cells with antibody complexes that bind to and cross-link CD3 and CD28 surface ligands on the CD4+ and CD8+ T cells.
[0165] In some embodiments, the CD4+ and CD8+ T cells are transduced with a plurality of viral vectors, wherein the viral vectors are lentiviral vectors. In some embodiments, the CD4+ and CD8+ T cells are transduced with the plurality of viral vectors about 24 hours following isolation from the PBMCs. In some embodiments, the CD4+ and CD8+ T cells are transduced with the plurality of viral vectors between about 20 hours to 22 hours, between about 22 hours to about 24 hours, or between about 24 hours to about 26 hours following isolation from the PBMCs. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the CD4+ and CD8+ T cell population is transduced with the viral vector that delivers genes to be screened.
[0166] In some embodiments, the viral vector comprises an expression cassette comprising a promoter operably linked to a barcoded gene. In some embodiments, the viral vector comprises a lentiviral-mediated delivery of an open reading frame (ORF) library (see Sack et al. Cell. 2018 Apr 5;173(2):499-514.e2, which is incorporated herein by reference). The term “barcode” or “barcode sequence” as used herein refers to a nucleotide sequence that corresponds to and allows for detection and/or identification of an expressed gene. The barcode typically comprises four or more nucleotides. In some embodiments, the barcode comprises 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, or 15 nucleotides. In some embodiments, the barcode comprises 8 to 15 nucleotides. As used herein, the terms “barcoded gene”, “barcoded ORF”, and the like refers to a nucleic acid that has an appended barcoded sequence, whether the barcode is linked directly to the 5’ or 3’ end of the ORF or separated by 1 or more nucleotides at the 5’ or 3’ end of the ORF. [0167] In some embodiments, the transduced CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene. In some embodiments, the drug-resistance gene comprises the puromycin N-acetyltransferase gene. In some embodiments, the transduced CD4+ and CD8+ T cells are selected for the expression of puromycin N-acetyltransferase by exposing the CD4+ and CD8+ T cells to puromycin. In some embodiments, the CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene about 72 hours following isolation from the PBMCs. In some embodiments, the CD4+ and CD8+ T cells are selected for the expression of a drug-resistance gene between about 68 to about 70 hours, about 70 to about 72 hours, or about 72 hours to about 74 hours following isolation from the PBMCs. In some embodiments, the transduced CD4+ and CD8+ T cells were cultured in media supplemented with puromycin throughout the culture period. In some embodiments, the viral vector further comprises nucleic acid encoding a selection gene (e.g., a fluorescent protein, GFP) that facilitates the further isolation or enrichment of transduced CD4+ and CD8+ T cells using flow cytometry. In some embodiments, the CD4+ and CD8+ T cells were cultured according to standard cell culture techniques commonly known in the art. In some embodiments, the CD4+ and CD8+ T-cells were either split or had media replaced to maintain a cell density of between about IxlO6 to about 2xl06 cells per mL about every 2 days. In some embodiments, the CD4+ and CD8+ T-cells were either split or had media replaced to maintain a cell density of between about IxlO6 to about 2xl06 cells per mL about every 3 days.
[0168] In some embodiments, a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to an immunosuppressive cellular environment. In some embodiments, the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
[0169] In some embodiments, a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to an adenosine-driven immunosuppressive cellular environment. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of between about 1 pM to about 64 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of between about 1 pM to about 4 pM, between about 4 pM to about 16 pM, or between about 16 pM to about 64 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 1 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 4 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 16 pM of adenosine. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 64 pM of adenosine.
[0170] In some embodiments, a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a TGF-P-driven immunosuppressive cellular environment. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of between about 0.25 pM to about 256 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of between about 0.25 pM to about 1 pM, between about 1 pM to about 4 pM, between about 4 pM to about 16 pM, between about 16 pM to about 64 pM, or between about 64 pM to about 256 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 0.25 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 1 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 4 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 16 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 64 pM of TGF-p. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of about 256 pM of TGF-p.
[0171] In some embodiments, a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a Treg-driven immunosuppressive cellular environment. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of between about 16:1 to about 1:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of between about 16:1 to about 8:1, about 8:1 to about 4:1, about 4:1 to about 2:1, or about 2:1 to about 1:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 16:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 8: 1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 4:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 2:1. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of Treg cells at a suppressor to effector ratio of about 1 : 1. In some embodiments, the Treg cells are differentiated induced Treg cells (iTreg). In some embodiments, the Treg cells are natural Treg cells (nTreg).
[0172] In some embodiments, a subset of transduced lymphocytes from the population of transduced lymphocytes are exposed to a macrophage-driven immunosuppressive cellular environment. In some embodiments, the subset of transduced lymphocytes is cultured in the presence of macrophage cells at a suppressor to effector ratio of about 1:1. In some embodiments, the macrophage cells are polarized macrophages.
[0173] In some embodiments, identification of the expressed gene is determined by PCR amplification of the gene and/or barcode sequence. In some embodiments, PCR is performed on genomic DNA (gDNA) obtained from the lymphocytes. In some embodiments, a reverse transcription step is performed to generate cDNA form the cell transcriptome and/or from an exogenous gene and barcode mRNA transcript. The amplified DNA products are then sequenced to identify an exogenous gene expressed in the isolated lymphocyte and/or to quantify the relative expression of an exogenous gene in a population of isolated lymphocytes. In some embodiments, the disclosed methods include RNA and/or DNA sequencing of lymphocytes using techniques that include, but are not limited to, whole transcriptome analysis, whole genome analysis, barcoded sequencing of whole or targeted regions of the genome, and combinations thereof. In some embodiments, RNA and/or DNA sequencing is performed in combination with proteome analysis. In some embodiments, the methods include detection of cell surface or intracellular proteins using, e.g., flow cytometry. In some embodiments, the methods comprise detection or identification the barcoded gene in combination with profiling additional molecular modalities using methods described in the art, including for example single-cell sequencing analysis (e.g., 10X Genomics Multiome platform), single-cell RNA-sequencing (scRNA-seq) (See, e.g., Haque et al. Genome Medicine, 9, Article number: 75 (2017); Hwang et al. Exp Mol Med. 2018 Aug 7;50(8):96), cell-hashing (See, e.g., Stoeckius et al. Genome Biol. 2018; 19: 224), Perturb-Seq. (See, e.g., Dixit et al. Cell. 2016 Dec 15; 167(7): 1853-1866x17), CROP-seq (See, e.g., Datbnger et al. Nat Methods. 2017 Mar;14(3):297- 301), CRISP-seq (See, e.g., Jaitin et al. Cell. 2016 Dec 15; 167(7): 1883-1896x15), Expanded CRISPR-compatible CITE-seq (ECCITE-seq) (See, e.g., Mimitou et al. Nat Methods. 2019 May;16(5):409-41), cellular indexing of transcriptomes and epitopes-seq (CITE-seq) (See, e.g., Stoeckius et al. Nat Methods. 2017 Sep;14(9):865-868), and overexpression-compatible cellular indexing of transcriptomes and epitopes by sequencing (OverCITE-seq) (See, e.g., Legut, Mateusz et al. Nature vol. 603,7902 (2022): 728-735). EXAMPLES
[0174] The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure.
Example 1. Methods to improve T cell function in an immunosuppressive environment.
[0175] The following example describes methods to conduct genome-scale screens of genes providing resistance to physiological drivers of immunosuppression.
Isolation and. culture of primary human cells
[0176] Leukopaks from de-identified healthy donors were collected by and purchased from StemcellPeripheral blood mononuclear cells (PBMC) were isolated from leukopaks using Lymphoprep (Stemcell) gradient centrifugation. For most assays, CD8+ and CD4+ were isolated sequentially from the same donor. First, CD8+ T-cells were isolated by magnetic positive selection using EasySep Human CD8 Positive Selection Kit II (Stemcell). Then, CD4+ T-cells were isolated from the resulting flowthrough by positive magnetic selection using EasySep Human CD4+ Positive Selection Kit II (Stemcell). The flowthrough from the CD4+ T cell isolation was used as a source of monocytes. Naive CD4+ T cells were isolated from PBMCs by magnetic selection using EasySep Human Naive CD4+ T Cell Isolation Kit. Regulatory T cells (Treg) were isolated using EasySep Human CD4+CD1271owCD25+ Regulatory T Cell Isolation Kit.
[0177] Immediately after isolation, naive CD4+ T cells, CD4+ or CD8+ T-cells were resuspended in T-cell Media, which consisted of Immunocult-XF T-cell Expansion Medium (Stemcell) supplemented with recombinant human IL-2 (Stemcell). Additionally, the media for naive CD4+ T cells was supplemented with TGF-|31 and retinoic acid for induced regulatory T cell (Treg) differentiation. Activation of naive CD4+ T cells, CD4+ or CD8+ T cells was performed with Immunocult Human CD3/CD28 T-cell Activator (Stemcell). Typically, CD4+ or CD8+ T cells were transduced with concentrated lentivirus 24 h post isolation. 72 h post isolation, lentivirally transduced CD4+ or CD8+ T cells were selected with puromycin. Every 2-3 days CD4+ and CD8+ T-cells were either split or had media replaced to maintain cell density of 1-2 x 106 cells per mL. Lentivirally transduced CD4+ or CD8+ T cells were maintained in media containing puromycin for the duration of culture. T- cells were used for phenotypic or functional assays fresh or cryopreserved in Bambanker Cell Freezing Media (Bulldog Bio).
[0178] The flowthrough enriched in monocytes was plated to achieve -90% confluence after 10 days of culture in RPMI 1640 media supplemented with FBS and cytokines M-CSF and GM-CSF (Stemcell). From day 5 after isolation, the culture media was further supplemented with at least of the following factors: dexamethasone, IL-4, IL-6, TGF-|31, TGF-|32, TGF-|33, IL- 10, IL- 113 (Stemcell). The culture conditions resulted in differentiation of monocytes into immunosuppressive polarized macrophages that were used in a co-culture with CD4+ and CD8+ T cells after 8 days post isolation.
Cell culture
[0179] HEK293FT cells were obtained from Thermo Fisher and cultured in DMEM supplemented with 10% Serum Plus-II (Thermo Fisher).
[0180] OV90 cells were obtained from American type culture collection (ATCC) and were cultured per ATCC recommendations. Endogenous Claudin 6 expression was verified by standard flow cytometry techniques using commercially available antibodies.
Lentivirus production
[0181] Lentivirus encoding the pooled ORF library was produced by co-transfecting a third- generation lentiviral transfer plasmid pool (cite 2nd application) together with the packaging plasmid psPAX2 and envelope plasmid pMD2.G into HEK293FT cells, using polyethyleneimine linear MW 25000 (Poly sciences). After 72 hours, the supernatants were harvested and filtered through a 0.45 pm Steriflip-HV filter (Millipore). The virus was concentrated using Lentivirus Precipitation Solution (Alstem). The concentrated lentivirus was then resuspended in T-cell Media containing IL-2 and stored at -80°C.
Flow cytometry for cell surface and intracellular markers
[0182] First, cells were harvested, washed with D-PBS and stained with LIVE/DEAD Violet cell viability dye for 5 minutes at room temperature in the dark, followed by surface antibody staining for 20 minutes on ice. After surface antibody staining (where applicable) the cells were washed with PBS and acquired on a flow cytometer or taken for intracellular staining. For intracellular staining, the cells were resuspended in FoxP3/Transcription Factor Fixation Reagent, (eBioscience). After resuspension in the fixation buffer, cells were incubated at room temperature in the dark for 1 hour. Following the incubation, the cells were washed twice in the FoxP3/Transcription Factor Permeabilization Buffer (eBioscience). After permeabilization, the cells were stained with the specific antibody or isotype control for 30 minutes in the dark at room temperature. Finally, the cells were washed twice in the appropriate permeabilization buffer and acquired on a flow cytometer. Gating was performed using appropriate isotype, fluorescence minus one and biological controls. Typically, 5,000- 10,000 live events were recorded per sample.
Pooled ORF library screening
[0183] For pooled ORF library screening, CD4+ and CD8+ T-cells were isolated from PBMC from three healthy donors and cultured separately for the first 9 days. The amount of lentivirus used for transduction was titrated to result in -40% transduction efficiency, to minimize the probability of multiple ORFs being introduced into a single cell. The cells were maintained in T-cell media containing puromycin and counted every 2-3 days to maintain cell density of 1-2 x 106 cells/mL. On day 10 after isolation, T-cells were harvested, counted, pooled at a 1:1 CD4:CD8 ratio and plated back in T cell media. An aliquot of cells representing l,000X coverage of the library was frozen down at this step to be used as a prestimulation control. For the following 4 days, an aliquot of pooled CD4:CD8 T cells was harvested and labelled with 5 |lM CellTrace Yellow (CTY) (Thermo Fisher) and stimulated with CD3/CD28 Activator (StemCell). Each assay was plated with a different immunosuppressive agent: adenosine (Sigma Aldrich), TGF-|31 (Stemcell), autologous induced regulatory T cells or autologous polarized macrophages. The cells were stained with LIVE/DEAD Violet cell viability dye (Thermo Fisher), CD4 and CD8 antibodies. CTYlow and CTYhigh cells (corresponding to the bottom 20% and top 20% of the distribution) were sorted using Sony MA900 or BD FACSAria cell sorter. Genomic DNA was isolated, and two rounds of PCR to amplify ORF barcodes and add Illumina adaptors were performed as described previously (Legut, M. et al. Nature 603, 728-735 (2022)). Pooled ORF screen analysis
[0184] Barcodes were mapped to the reference library after adaptor trimming with cutadapt using bowtie. All subsequent analyses were performed in RStudio with R. To calculate individual barcode enrichment, barcode counts were normalized to the total number of reads per sample (with pseudocount added) and log2 transformed. Statistical analysis on barcode enrichment was performed using MAGeCK, comparing CTY1OW samples to corresponding CTYhlgh samples for each immunosuppressive conditions, using three donors and two cell types (CD4+, CD8+) as replicates.
Statistical analysis
[0185] Data between two groups were compared using a two-tailed unpaired Student’s t-test or the Mann- Whitney test as appropriate for the type of data (depending on normality of the distribution). Unless otherwise indicated, a P-value less than or equal to 0.05 was considered statistically significant for all analyses, and not corrected for multiple comparisons. In cases where multiple comparison corrections were necessary, we adjusted the P-value using the Benjamini-Hochberg method. All group results are represented as mean ± s.e.m, if not stated otherwise. Statistical analyses were performed in Prism (GraphPad) and RStudio (Rstudio PBC). Flow cytometry data was analyzed using FlowJo 10.7.1 (Treestar).
Example 2. In vitro assays to recapitulate immunosuppressive cellular environments and methods to quantify T cell proliferation.
[0186] Four key mediators of TME immunosuppression were identified. These mediators of TME immunosuppression comprise adenosine, TGF-P, regulatory T cells, and immunosuppressive macrophages. The following example establishes methods to assess T cell proliferation following exposure to these mediators of TME immunosuppression.
[0187] To assess whether adenosine and TGF-P would produce an immunosuppressive environment in vitro, T cells were cultured in the presence varying concentrations of either agent while monitoring their proliferation and viability. As shown in FIG. 1A, CD4+ and CD8+ T cells experience a dose-dependent suppression of proliferation when cultured in the presence of adenosine. Moreover, this dose-dependent suppression of proliferation was not a result of a reduction in cell viability (FIG. IB). A similar dose dependent inhibition of proliferation was observed when CD4+ and CD8+ T cells were cultured in the presence of TGF-P (FIG. 2A). The suppression of T cell proliferation in the presence of TGF-P was also not a result of a reduction in T cell viability (FIG. 2B). These results demonstrate that culturing T cells in the presence of either adenosine or TGF-P can recapitulate an immunosuppressive environment in vitro.
[0188] Regulatory T cells were also identified as contributors to immunosuppressive environments. In order to establish an in vitro culture system recapitulating this immunosuppressive effect, natural Tregs (nTregs) were successfully isolated and expanded and differentiated induced Tregs (iTregs) were successfully differentiated from naive CD4+ T cells. Both nTregs and iTregs showed robust expression of the key transcription factor FoxP3, were able to suppress proliferation of CD4+ and CD8+ T cells in a dose-dependent manner and expanded well ex vivo (FIGs. 3A-3E).
[0189] To assess the immunosuppressive effect of co-culturing T cells with macrophages, human macrophages were successfully differentiated from peripheral blood monocytes and polarized towards an immunosuppressive phenotype. The polarized macrophages showed phenotypic hallmarks of immunosuppressive M2-like macrophages, such as a decreased expression of CD64 and an increased expression of CD206 and PD-L1 (FIG. 4A). The polarized macrophages were also capable of suppressing T cell proliferation across a range of polarization conditions (dexamethasone, IL-4, IL-6, TGF-pi, TGF-P2, TGF-P3, IL-10, IL-ip) (FIG. 4B).
[0190] In order to identify immunosuppressive resistance genes allowing T cells to proliferate despite exposure to immunosuppression, a proliferation assay was developed utilizing the cell-labeling dye CTY. This dye enabled the quantification of T cell divisions, wherein every cell division resulted in a reduction in the amount of dye in the daughter cells of about 50% compared to the parent cell as measured by flow cytometry. The frequency of the most proliferative cells (that is, cells with lowest amount of the CTY dye) correlates well with gold- standard measures of T cell proliferation such as the proliferation index (FIG. 5A) and the absolute cell count (FIG. 5B).
Example 3. Methods to improve T cell function in an immunosuppressive environment.
[0191] The overexpression of a barcoded library of genes in primary T cells was previously utilized to identify key regulators of T cell proliferation and other effector functions (Legut, M. et al. Nature 603, 728-735 (2022)). The following example provides substantial enhancements to this screening approach to better mimic the complex cellular environment found in clinical settings. These improvements have led to higher data quality and a robust discovery of immunosuppressive resistance genes (FIG. 6). [0192] The method comprises the co-culture of stimulated, library-transduced T cells with immunosuppressive factors. This includes the incorporation of autologous immunosuppressive cell types and culturing both CD4+ and CD8+ T cells in the same assay. The interaction between CD4+ and CD8+ T cells is crucial for the clinical efficacy of cell therapy products. Therefore, the immunosuppressive resistance genes identified in the context of CD4+ and CD8+ co-culture may hold more clinical relevance than those identified in CD4+ and CD8+ T cells when tested separately (Sommermeyer, D. el al. Leukemia 30, 492- 500 (2016)).
[0193] Importantly, the supraphysiological conditions for T cell stimulation in previous approaches made it impossible to measure the effect of immunosuppression. For example, cells were previously cultured in the presence of 400 lU/mL of IL-2 to drive T cell proliferation in culture. This level of IL-2 stimulation could have prevented the immunosuppressive activities of adenosine, TGF-P, regulatory T cells, or immunosuppressive macrophages described above. Consequently, conditions were established to both activate T cells in culture while enabling the controlled immunosuppressive effects of adenosine, TGF- P, regulatory T cells, and immunosuppressive macrophages in a physiological manner (FIGs. 7A, 7B). Such conditions include the reduction in IL-2 supplementation during culture from a concentration of 400 lU/mL to a concentration of 50 lU/mL.
[0194] The methods described herein facilitated the execution of multiple screens per healthy T cell donor (FIGs. 8A, 8B). Coverage is a critical factor for robust data capture and maintaining reliable signal to noise ratios in pooled genomic screens (Sanjana, N. E. Anal. Biochem. 532, 95-99 (2017)). Furthermore, the capability to conduct multiple screens from the same donor enables the identification of immunosuppressive resistance genes that protect T cells from various forms of immunosuppression.
[0195] The immunosuppression assays described in Example 2 were effectively scaled to enable genome-scale screens. A total of 12 screens (3 healthy donors, 4 immunosuppressive mechanisms) comprising co-culture of both CD4+ and CD8+ T cells were conducted. Across all screens, a robust suppression of T cell proliferation was observed (FIG. 9). The modifications to the previous platform described above led to a marked improvement in data quality, as measured by increased barcode recovery in the most proliferative T cell populations (FIG. 10).
[0196] In total, these genome-scale screens uncovered 800 genes providing resistance to adenosine immunosuppression (Table 1), 722 genes providing resistance to TGF-P immunosuppression (Table 2), 707 genes providing resistance to Treg immunosuppression (Table 3), and 909 genes providing resistance to macrophage immunosuppression (Table 4). Additionally, 379 of the identified genes provided resistance to 2 different modes of immunosuppression (Table 5) and 51 of the identified genes provided resistance to 3 or more modes of immunosuppression (Table 6). Out of the 51 genes, one gene (LTBR) has been shown to improve CAR and TCR T cell proliferation and other effector functions (Legut, M. et al. Nature 603, 728-735 (2022)) while another gene (PDE4A) has been shown to improve CAR T cells resistance to adenosine, but not other forms of immunosuppression (Schmetterer, K. G. et al. Front. Immunol. 10, 1790 (2019)).
Table 1. Genes providing resistance to adenosine-driven immunosuppression. ANKRD45 236 FKBP14 436 NAE1 636 SNX5 340 IL13RA2 540 PSMC4 740 UGDH
Table 2. Genes providing resistance to TGF-P-driven immunosuppression.
Table 3. Genes providing resistance to Treg-driven immunosuppression.
Table 4. Genes providing resistance to macrophage-driven immunosuppression. Ill CDC42EP4 361 IL13RA1 611 PSMC4 861 WDR37
Table 5. Genes providing resistance to two immunosuppressive cellular environments.
environments. Conclusions
[0197] Overall, substantial improvements were made to the previous immunosuppressive resistance gene platforms, which have enabled, for the first time, identification of key genetic factors protecting T cells from TME immunosuppression. These methods enabled the identification of 800 genes providing resistance to adenosine immunosuppression, 722 genes providing resistance to TGF-P immunosuppression, 707 genes providing resistance to Treg immunosuppression and 909 genes providing resistance to macrophage immunosuppression, 379 genes providing resistance to 2 different modes of immunosuppression and 51 of the genes providing resistance to 3 or more modes of immunosuppression when overexpressed in primary CD4+ and CD8+ T cells.
Example 4. Methods and compositions for improving T cell tumor-killing capacity in an immunosuppressive environment.
[0198] The genome-scale overexpression screens described above were used to identify immunosuppressive resistance genes which, when overexpressed in T cells, enhanced T cell expansion by protecting them from various forms of tumor immunosuppression. The following example provides further characterization of the function of these immunosuppression resistance genes in the context of cancer, particularly in primary T cells comprising CARs targeting solid tumor antigens.
Individual overexpression of immunosuppressive resistance genes in CAR-T cells [0199] First, selected immunosuppressive resistance genes identified by genome-scale screens in the immunosuppressive contexts were cloned from the screening library or synthesized. For expression in T cells, the immunosuppressive resistance genes were cloned into a lentiviral or retroviral vector which typically encoded a marker gene for selection of transduced cells. Example selection markers include puromycin N-acetyltransferase, blasticidin-S deaminase, truncated nerve growth factor receptor, or truncated epidermal growth factor receptor. In some instances, the vector also encoded a CAR, for instance a CAR targeting mesothelin, HER2, or claudin 6 (CEDN6) as described, for example in WO 2015/090230 Al, WO 2017/079694 A2, or US 2022/0184119 Al, all of which are herein incorporated by reference in their entireties. For benchmarking purposes, in some instances the vector encoded a previously described gene in the context of CAR T cell armoring, such as cJUN, membrane bound IE15 or dominant negative TGF-P receptor 2 instead of immunosuppressive resistance genes. CAR T cell armoring standards are described, for example, in WO 2023/172514 Al, US 2022/0307039 Al, and US 2023/0220343 Al, all of which are herein incorporated by reference in their entireties. For vectors encoding more than one transgene, the transgenes were separated with self-cleaving 2A peptides such as P2A or T2A. For lentiviral vectors, the expression was driven by the short EF-loc or SFFV promoters. For retroviral vectors, the expression was driven by a MoMLV-based promoter. [0200] The immunosuppressive resistance genes were delivered to activated primary human T cells (total CD3+, CD4+, CD8+ or gamma delta T cells). To achieve co-expression of the immunosuppressive resistance gene with a CAR, the cells were either transduced with a tricistronic vector encoding the CAR, immunosuppressive resistance gene and a marker gene (see, e.g., FIGs. 15-19), or co-transduced with two bicistronic vectors, one encoding a CAR and a marker gene, and the other encoding a immunosuppressive resistance gene and another marker gene (see, e.g., FIGs. 11-14B). The sequences of immunosuppressive resistance genes are listed in the Sequence Table below.
Tumor-killing assay
[0201] Following selection based on the expression of marker genes, the transduced cells were characterized in terms of their cytotoxic activity against cancer cells expressing the CAR target. In some assays, the co-culture also included immunosuppressive factors such as polarized macrophages, regulatory T cells, adenosine or TGF-|3. Macrophages and regulatory T cells were generated as described for the genome-scale immunosuppressive resistance gene screens (Example 1).
[0202] To understand how the selected immunosuppressive resistance genes affect direct tumor killing of modified CAR-T cells, a three-component co-culture system was applied to model the tumor microenvironment in vitro'. Three-component co-cultures included CAR T cells expressing immunosuppressive resistance genes, tumor cells, and components of the immunosuppressive microenvironment (e.g., macrophages, Tregs, adenosine or TGF-|3). [0203] First, the effect of overexpression of immunosuppressive resistance genes on CAR-T tumor killing capacity was tested in a regulatory T cell (Treg) immunosuppressive environment in vitro. CD4+ and CD8+ T cells co-expressing a HER-2-specific CAR and an indicated immunosuppressive resistance gene were co-incubated with GFP-engineered HER2+ cancer cell lines for up to 96 h at 1:8 T cells to cancer cells ratio. Tested immunosuppressive resistance genes include FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL, the nucleotide and amino acid sequences of which are listed in the Sequence Table below. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to the value observed in unmodified CAR T cells in the absence of immunosuppression (100%). As shown in FIG. 11, ectopic expression of these immunosuppressive resistance genes resulted in increased tumor killing modified CAR-T cells in the presence of Treg-mediated immunosuppression compared to unmodified CAR-T cell controls, suggesting an ability for these genes to reduce the negative effect of immunosuppression on cancer killing.
[0204] Using the same tumor killing assay, immunosuppressive resistance genes COPZ2, GNL3, LIMA1, and LTBR, the nucleotide and amino acid sequences of which are listed in the Sequence Table below, were next tested for their effects on CAR-T tumor-killing capacity in adenosine immunosuppression in vitro. As shown in FIG. 12, ectopic expression of these immunosuppressive resistance genes similarly resulted in increased tumor killing modified CAR-T cells in the presence of adenosine immunosuppression compared to unmodified CAR-T cell controls. Next, immunosuppressive resistance genes CD47, CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC STK11, YBX2, and ZTBTB46, the nucleotide and amino acid sequences of which are listed in the Sequence Table below, were tested for their effects on CAR-T tumor-killing capacity in macrophage- mediated immunosuppression in vitro with the same tumor-killing assay. As shown in FIG. 13, ectopic expression of these immunosuppressive resistance genes also resulted in increased tumor killing capacity of modified CAR-T cells in the presence of macrophage-mediated immunosuppression compared to unmodified CAR-T cell controls.
[0205] Taken together, these results show that, across the range of immunosuppressive factors, the immunosuppressive resistance genes selected in the genome-wide screens reduce the negative effect of immunosuppression on cancer cell killing, in some cases resulting in an even stronger killing than unmodified CAR T cells in absence of immunosuppression.
T cell exhaustion analysis
[0206] In addition to immunosuppression, T cell exhaustion poses another major limitation to the efficacy of solid tumor cell therapies. To understand the impact of select immunosuppressive resistance genes on T cell exhaustion, a similar tumor-killing assay was performed wherein a T cells comprising a CAR targeting a solid tumor antigen were repeatedly challenged with cancer cells to recapitulate the process leading to T cell exhaustion in patients. CD4+ and CD8+ T cells co-expressing a HER2-specific CAR and ZBTB46, the nucleotide and amino acid sequence of which is listed in the Sequence Table below, or a control irrelevant gene tNGFR were co-incubated with GFP-engineered HER2+ cancer cell lines at 1:4 T cells to cancer cells ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. Killing was normalized to account for the presence of a specified immunosuppressive factor on cancer cell growth. As shown in FIG. 14A, ectopic expression of ZTBTB46 resulted in maintenance of stronger cytotoxic activity against cancer cells throughout the length of the cancer cell challenge in a normal culture background, while FIG. 14B similarly shows increased maintenance of tumor killing capacity of modified CAR-T cells in a TGF- P immunosuppressive culture environment.
[0207] Next, similar experiments were performed to test the effect of the immunosuppressive resistance gene LTBR (DNA sequence SEQ ID NO: 19; amino acid sequence SEQ ID NO: 38) on T cell exhaustion when challenged with cancer cells in vitro. CD4+ and CD8+ T cells co-expressing a HER2- specific CAR and LTBR were co-incubated with GFP-engineered HER2+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. When CAR T cells stopped specifically killing cancer cells, they were removed from the subsequent rounds of cancer cell challenge. These tests were performed in parallel with clinically developed, benchmark CAR-T armoring genes cJUN, membrane-bound IL 15 (mbIL15) or dominantnegative TGF- receptor 2 (TGFBR2dn) to determine the relative efficacy of LTBR in preventing T cell exhaustion in a cancer-killing context. As shown in FIG. 15, LTBR coexpression with a HER2 CAR utilizing a CD28-based costimulatory domain enabled the T cells to clear multiple cancer challenges, to a much stronger extent than the other benchmark armoring genes.
[0208] Similar tests on the effect of LTBR on T cell exhaustion were performed using CLDN6-28z y8 CAR-T cells. Here, CAR T cells co-expressing a control gene tNGFR (“Unmodified CAR”) or armoring genes LTBR, mbIL15 or TGF RIIDN were co-incubated with GFP+ OV90 ovarian cancer cells at 1:1 effectortarget ratio for 72-96 hours. After each round of co-incubation, T cells were harvested and added to fresh cancer cells. Killing was determined at endpoint of each co-incubation by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone. As shown in FIG. 21, armoring with LTBR enhanced tumor killing capacity of CLDN6-28z y8 CAR-T cells in a repeated tumor challenge model in vitro, further establishing the ability of LTBR to prevent T cell exhaustion beyond benchmark armoring genes.
[0209] Further still, the effect of LTBR on T cell exhaustion were performed using CLDN6 cx(3 CAR T cells, wherein the CAR is as described in Feucht et al., Nat. Med., (2018) 25(l):82-88, the content of which is herein incorporated by reference in its entirety. Here, CAR T cells, co-expressing a control gene tEGFR (“Unmodified CAR”) or armoring gene LTBR were co-incubated with GFP+ SKOV3 ovarian cancer cells at 1:16 effector: target ratio for up to 140 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone. As shown in FIG. 23, armoring with LTBR enhanced tumor killing capacity of CLDN6 cx|3 CAR T cells relative to control CAR T cells, further establishing the ability of LTBR to enhance CAR T cell tumor killing capacity.
[0210] To confirm the function of LTBR in preventing T cell exhaustion, a tumor killing assay was performed comparing the effect of LTBR and negative control LTBR-del on CLDN6-28z cx[3 CAR T cell efficacy in vitro. LTBR-del is a non-functional, truncated version of LTBR lacking the intracellular signaling domain, resulting in an inability to transmit signaling even when the extracellular part binds a ligand. CLDN6-28z cx[3 CAR T cells, co-expressing a control gene tEGFR (“Unmodified CAR”), armoring gene LTBR, or LTBR-del, were co-incubated with GFP+ OV90 ovarian cancer cells at 1:32 effector:target ratio for up to 110 hours. Killing was determined by comparing the GFP signal in CAR T treated wells to wells containing cancer cells alone. As shown in FIG. 22, LTBR enhanced CLDN6-28z cx[3 CAR efficacy in vitro, which was abolished in LTBR-del expressing CAR T cells. This result confirmed that functional LTBR enhanced CAR T cell tumor killing capacity in vitro.
[0211] Serial in vitro cancer challenges were then repeated in the presence of Tregs to determine the efficacy of LTBR in preventing T cell exhaustion in an immunosuppressive environment. As shown in FIG. 16, LTBR-expressing CAR T cells were also able to withstand immunosuppression, in particular mediated by Tregs, in the repeated cancer challenge context, notably outperforming CAR-T cells expressing benchmark armoring genes cJUN, mbIL15, and TGFBR2dn at the second cancer cell challenge.
[0212] To confirm that the benefits of LTBR expression on T cell function were preserved across different antigen targets, Claudin-6 (CLDN6)-targeting CAR-T cells utilizing a 4- IBB costimulatory domain were co-expressed with LTBR and similarly tested for tumor killing capacity against the CLDN6-high PAI and CLDN6-med OV90 ovarian cancer cell lines using a similar serial challenge assay as described above. Briefly, CD4+ and CD8+ T cells co-expressing a CLDN6-specific CAR and LTBR or a control irrelevant gene were co- incubated with GFP-engineered CLDN6+ cancer cell lines at 1:1 T cells to cancer cells ratio. Every 3-4 days T cells were challenged with fresh cancer cells, as indicated. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. As shown in FIG. 17, LTBR expression resulted in a similar improvement of cytotoxic activity in the repeated cancer challenge assay, a proxy for resistance to exhaustion, in the context of a CLDN6 CAR utilizing a 4- IBB costimulatory domain in the context of ovarian cancer lines expressing different levels of the target antigen.
[0213] The effect of LTBR expression on T cell function was tested in y8 T cells to ensure benefits are not restricted to cx[3 T cells. Here, y8 T cells co-expressing a CLDN6-specific CAR and LTBR or a control irrelevant gene were co-incubated with GFP-engineered CLDN6+ cancer cell line OV90. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. After 96 h of co-incubation, T cells were harvested and stained in the presence of counting beads to determine their expansion and exhaustion. Exhausted cells are defined as LAG3+ TIM3+. These experiments showed that the extent of tumor-killing was increased with LTBR expression (FIG. 18A), while expansion of CLDN6-targeting y8 CAR-T cells was also increased with LTBR expression (FIG. 18B), and exhaustion was decreased in the LTBR-expressing T cells (FIG. 18C). These results confirmed that LTBR-mediated improvement to CAR T cell function extends beyond cx[3 T cells to y8 T cells as well.
[0214] The ability of CLDN6-targeting y8 CAR-T cells to kill antigen-low cancer cells was tested. Here, y8 T cells co-expressing a CLDN6-specific CAR and LTBR or a control irrelevant gene were co-incubated with GFP-engineered CLDN6low cancer cell line SKOV3 for 88 h at 1:4 T cell to cancer cell ratio. Killing was determined by comparing the GFP signal in wells with T cells and cancer cells to cancer cells alone. As shown in FIG. 19, CAR y8 T cells expressing LTBR were also able to clear antigen-low ovarian cancer cells which were poorly targeted by unmodified CAR T cells. This result suggests that ectopic LTBR expression in lymphocytes can potentially increase the number of patients amendable to this therapeutic intervention, since T cell efficacy at a lower cancer-antigen threshold would mean more patients qualify for this treatment. Furthermore, LTBR expression can potentially lead to more durable therapeutic responses, as most CAR-based therapies are only able to target high antigen-expression cancer cells, thereby leading to incomplete or inefficient tumor killing capacities.
CAR-T Animal Studies
[0215] Finally, the ability of CLDN6-targeting aP CAR-T cells to kill antigen-expressing tumor cells in vivo was tested. To generate CAR-T cells for animal studies, aP T cells were activated by CD3/CD28 stimulation on day 0. On day 2 T cells were transduced with gamma retroviral particles carrying a Claudin 6-targeting 28z CAR transgene followed by a 2A sequence and a modifier gene. Cells were expanded until day 7, when transduction efficiency was measured, and cells were cryopreserved in standard cryopreservation media.
[0216] To form tumors for experimental analysis, OV90 ovarian cancer cells were obtained from American type culture collection (ATCC) and were cultured per ATCC recommendations until ready for use in mouse models. Endogenous Claudin 6 expression was verified by standard flow cytometry techniques using commercially available antibodies. After OV90 cells were expanded in vitro, 6-8 week old, female, NOD.Cg-
Prkdcscld H2rgtml wjl/SzJ (NSG) mice were subcutaneously inoculated with 5xl06 OV90 cells to form tumors. Tumor volumes were measured twice weekly by caliper. Once tumors reached 150 mm3 to 190 mm3, mice were assigned to treatment groups, in order to achieve tumor volume uniformity. Following grouping, mice received CAR T cells at various doses. [0217] Once tumors reached proper volume, the NSG mice were injected intravenously with 1.25 x 106 CAR T cells co-expressing a control gene tEGFR (n = 4) or LTBR (n =5), or donor-matched T cells not expressing any CAR (“No CAR”, n=13). Following T cell injection, survival of the animals was monitored daily. Animals were removed from the study and euthanized upon reaching a tumor burden of over 2,000 mm3 or another humane endpoint. * p<0.05 (log-rank test). After reaching the experimental endpoint, explanted tumors were weighed and dissociated by enzymatic digestion. Equal numbers of cells were taken from each digested sample and were processed for flow cytometry analysis using standard techniques. To quantify cells in each sample, a fixed number of quantification beads were added. Tumor cells were identified as a human HLA-ABC positive and CD4/CD8 negative population. T cells were identified as human HLA-ABC and CD4/CD8 positive populations.
[0218] As shown in FIG. 20A, CAR-T cells co-expressing LTBR showed enhanced survival relative to both no CAR T cell control and CAR-T cells co-expressing the tEGFR control gene. No significant changes in mouse body weight were observed in the CAR-T + LTBR group relative to either control group (FIG. 20B).
[0219] At the experimental endpoint, matched tumor samples were explanted and processed to assess tumor infiltration by human T cells. As shown in FIG. 20C, OV90 tumors in mice treated with CAR-T cells co-expressing LTBR exhibited increased tumor infiltration by human T cells relative to CAR_T cells co-expressing the control tEGFR gene. Finally, as shown in FIG. 20D, all in vivo tumor cells exhibited decreased CLDN6 levels relative to OV90 in vitro control cells, demonstrating that CAR-T cells co-expressing LTBR have increased ability to target antigen-low cancer cells in vivo relative to controls.
[0220] Overall, these results show that overexpression of certain human genes in T cells results not only in enhancement of T cell proliferation in the context of various forms of immunosuppression but also in enhancement of direct cancer cell killing. In addition to mitigating the negative effects of tumor immunosuppressive microenvironment, the described genes also mitigate T cell exhaustion by maintaining strong cytotoxic activity through multiple rounds of cancer challenge in different antigen, tumor type and CAR architecture context. The utility of described immunosuppressive resistance genes extends beyond cx[3 T cells as they show similar effects in y8 T cells.
SEQUENCE TABLE
Ill
EXEMPLARY EMBODIMENTS
Embodiment 1. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
Embodiment 2. The modified lymphocyte of Embodiment 1, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 3. The modified lymphocyte of Embodiment 1 or Embodiment 2, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 4. The modified lymphocyte of any one of Embodiments 1-3, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
Embodiment 5. The modified lymphocyte of any one of Embodiments 1-3, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, ADARB1, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, D0K5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EX01, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FM05, FNDC9, F0XJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
Embodiment 6. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, C0PZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
Embodiment 7. The modified lymphocyte of Embodiment 6, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 8. The modified lymphocyte of Embodiment 6 or Embodiment 7, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 9. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the modified lymphocyte has increased proliferation and/or increased effector function in in vivo or in vitro tumor killing assays relative to an unmodified lymphocyte.
Embodiment 10. The modified lymphocyte of Embodiment 9, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding LTBR.
Embodiment 11. The modified lymphocyte of Embodiment 9 or Embodiment 10, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding LTBR.
Embodiment 13. The modified lymphocyte of any one of Embodiments 6-8, wherein the increased level of the immunosuppressive resistance gene results in increased tumor cell killing of the modified lymphocyte in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene.
Embodiment 14. The modified lymphocyte of any one of Embodiments 1-13, wherein the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6. Embodiment 15. The modified lymphocyte of any one of Embodiments 1-14, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor
(TCR).
Embodiment 16. The modified lymphocyte of Embodiment 15, wherein the CAR or the TCR binds to a tumor antigen.
Embodiment 17. The modified lymphocyte of Embodiment 15 or Embodiment 16, wherein the increased level of the immunosuppressive resistance gene LTBR results in increased killing of cells expressing a low level of antigen in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene, wherein the antigen is bound by the CAR or the TCR.
Embodiment 18. The modified lymphocyte of Embodiment 17, wherein the tumor killing assay is an in vitro tumor killing assay.
Embodiment 19. The modified lymphocyte of any one of Embodiments 15-18, wherein the CAR or the TCR binds to HER2 or Claudin-6 (CLDN6).
Embodiment 20. The modified lymphocyte of any one of Embodiments 15-19, wherein the modified lymphocyte further comprises an expression cassette comprising a nucleic acid encoding the CAR or the TCR.
Embodiment 21. The modified lymphocyte of Embodiment 20, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in the same expression cassette.
Embodiment 22. The modified lymphocyte of Embodiment 20, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in separate expression cassettes.
Embodiment 23. The modified lymphocyte of any one of Embodiments 1-22, wherein exhaustion of the modified lymphocyte is reduced compared to a lymphocyte that does not express the immunosuppressive resistance gene.
Embodiment 24. The modified lymphocyte of any one of Embodiments 1-23, wherein the modified lymphocyte maintains the ability to kill tumor cells expressing a tumor antigen following at least two exposures to the tumor cells.
Embodiment 25. The modified lymphocyte of any one of Embodiments 1-24, wherein the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
Embodiment 26. The modified lymphocyte of any one of Embodiments 1-25, wherein the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs). Embodiment 27. The modified lymphocyte of any one of Embodiments 1-26, wherein the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
Embodiment 28. The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
Embodiment 29. The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
Embodiment 30. The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a TCRy8+ lymphocyte.
Embodiment 31. The modified lymphocyte of Embodiment 30, wherein the modified lymphocyte is a TCRy8+ lymphocyte and is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR.
Embodiment 32. The modified lymphocyte of Embodiment 31, wherein the increased level of the immunosuppressive resistance gene LTBR results in increased killing of antigen-low cancer cells in tumor killing assays compared to an unmodified lymphocyte.
Embodiment 33. The modified lymphocyte of Embodiment 32, wherein the tumor killing assay is an in vitro tumor killing assay.
Embodiment 34. The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
Embodiment 35. The modified lymphocyte of any one of Embodiments 1-27, wherein the modified lymphocyte is a regulatory T cell.
Embodiment 36. The modified lymphocyte of any one of Embodiments 1-35, wherein the modified lymphocyte is a human lymphocyte.
Embodiment 37. The modified lymphocyte of any one of Embodiments 1-36, wherein the modified lymphocyte is an autologous lymphocyte.
Embodiment 38. The modified lymphocyte of any one of Embodiments 3-37, wherein the modified lymphocyte comprises a vector comprising the expression cassette.
Embodiment 39. The modified lymphocyte of any one of Embodiments 3-38, wherein the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
Embodiment 40. The modified lymphocyte of Embodiment 39, wherein the promoter is a ubiquitous promoter. Embodiment 41. The modified lymphocyte of Embodiment 40, wherein the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate- early enhancer and chicken beta-actin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
Embodiment 42. The modified lymphocyte of Embodiment 39, wherein the promoter is an inducible promoter.
Embodiment 43. The modified lymphocyte of any one of Embodiments 39-42, wherein the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
Embodiment 44. The modified lymphocyte of any one of Embodiments 39-43, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte.
Embodiment 45. The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene.
Embodiment 46. The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus.
Embodiment 47. The modified lymphocyte of any one of Embodiments 39-44, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
Embodiment 48. A vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof.
Embodiment 49. The vector of Embodiment 48, wherein the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 50. The vector of Embodiment 48 or Embodiment 49, further comprising nucleic acid encoding a therapeutic protein.
Embodiment 51. The vector of Embodiment 50, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Embodiment 52. The vector of Embodiment 51, wherein the nucleic acid encoding the
CAR or TCR are included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 53. The vector of Embodiment 51, wherein the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or TCR are included in a second expression cassette.
Embodiment 54. The vector of any one of Embodiments 48-53, wherein the vector is a viral vector.
Embodiment 55. The vector of any one of Embodiments 48-54, wherein the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
Embodiment 56. The vector of Embodiment 55, the vector is an episomal or nonintegrating vector.
Embodiment 57. The vector of Embodiment 56, wherein the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
Embodiment 58. The vector of any one of Embodiments 48-53, wherein the vector is a non-viral vector.
Embodiment 59. The vector of Embodiment 58, wherein the non-viral vector is a plasmid.
Embodiment 60. The vector of any one of Embodiments 48-59, wherein the vector further comprises nucleic acid encoding a drug-resistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
Embodiment 61. A modified lymphocyte comprising one or more of the vectors as defined in any one of Embodiments 48-60.
Embodiment 62. A composition comprising the modified lymphocyte as defined in any one of Embodiments 1-47.
Embodiment 63. The composition of Embodiment 62, wherein the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
Embodiment 64. A method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the vector of any one of Embodiments 48-60.
Embodiment 65. The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a tumor microenvironment. Embodiment 66. The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
Embodiment 67. The method of Embodiment 66, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, ADARB1, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP6V1C1, ATP6V1D, ATPAF1, ATRAID, ATRIP, AUTS2, AZI2, B3GNT9, BAIAP2, BAIAP2L2, BAP1, BATE, BCAT1, BCKDHA, BCL2L13, BCL2L2, BLMH, BMPER, BOD1, BPIFA1, BSG, BSPRY, BTC, BTD, BTRC, C18orf54, C1QTNF12, C1QTNF4, C3orf38, C6orf223, C8orf76, C9orfll6, CALCOCO1, CALHM1, CAT, CAVIN1, CAVIN4, CBWD1, CCDC113, CCDC121, CCDC14, CCDC174, CCDC86, CCDC93, CCIN, CCN2, CCN3, CCR6, CCSAP, CCT7, CD19, CD1B, CD36, CD46, CD86, CD96, CDC14A, CDH1, CDK2AP1, CDK7, CELA2B, CEP97, CFAP46, CFAP47, CHAMP1, CHMP2B, CHST8, CKB, CLEC11A, CLEC4M, CLEC9A, CLPB, CLPSL1, CLSTN1, CLUL1, CLYBL, CNDP2, CNTROB, COA6, COL15A1, COL21A1, COL22A1, COLQ, COMMD5, COPZ2, CORO6, COX11, CPA1, CPSF6, CPT2, CRB3, CREB3L2, CREM, CRHR2, CRTC2, CRYGD, CSK, CTBP2, CTNNA2, CTSS, CX3CR1, CYP20A1, CYP2C9, DADI, DAXX, DBF4, DCDC2, DCLK2, DCTN3, DCX, DDB1, DDHD1, DDO, DDOST, DDX21, DDX56, DEGS2, DEPDC7, DFFB, DIXDC1, DLK1, DNAI2, DNAJC16, DPPA4, DPYSL3, DPYSL4, DRG2, DUT, E2F7, EBNA1BP2, ECHDC1, ECHDC3, ECU, ECRG4, EDC3, EFCAB1, EFCAB12, EFCAB13, EFEMP2, EGR2, EIF2A, EIF4G3, EMP1, ENPP3, EPHA4, EPHB6, EPX, ERCC2, ERN2, ERV3-1, ESRP1, ETNK2, EVI2B, EXO1, EXTL3, EZH2, F10, F13B, F2, FAF2, FAM110A, FAM13C, FAM161B, FAM181A, FAM200A, FAM71A, FAM98A, FANCB, FATE1, FBXL13, FBXL16, FBXL20, FCRLB, FEM1C, FEV, FGF22, FGF3, FKBP14, FKBP5, FKBPL, FLOT1, FLVCR1, FLYWCH1, FMN1, FMO5, FOSB, FOXJ1, FOXS1, FPGT, FRMD5, FUT8, G6PC3, GAB3, GABRA5, GABRB2, GABRQ, GAD2, GALNT7, GAN, GAPDHS, GAPVD1, GBP4, GCNA, GDF6, GDF9, GDPD5, GET1, GET4, GGA1, GGA2, GGTLC1, GIMAP8, GKAP1, GLRB, GLT6D1, GLTPD2, GNAI2, GNL3, G0RASP1, GORASP2, GOSR1, GPAA1, GPC5, GPR119, GPR37, GPR65, GPRASP2, GPRC5C, GPT2, GPX1, GRHL2, GRID1, GRINA, GSDME, GTF2H1, GTF2I, GTPBP2, GUSB, GYPA, H2BC4, HABP4, HACD3, HAGH, HAND2, HARS2, HAUS7, HAX1, HDAC9, HDHD3, HENMT1, HEPACAM2, HHEX, HIBCH, HID1, HK1, HLA-C, HLCS, HNF4A, HNRNPAB, HNRNPD, H0XA11, H0XD8, HP1BP3, HPS3, HPS4, HSDL1, HSP90B1, HTR1F, HTRA4, HVCN1, IBSP, IDS, IFNA8, IFNAR2, IFNGR1, IFNLR1, IGFBP1, IGKV1D-17, IHO1, IK, IL10RA, IL12RB2, IL13RA2, IL17RE, IL36G, IMPDH2, INF2, INKAI, INTS5, IP6K2, IPO5, IQCA1, IREB2, JAGN1, JMJD8, KAT14, KAT7, KAZALD1, KCNAB3, KCNG3, KCNJ14, KCNK12, KCNK4, KCNK9, KCNMB2, KCNV1, KCTD4, KHDRBS2, KHDRBS3, KIAA0930, KIF7, KIFC3, KLHL14, KLHL2, KLHL8, KREMEN2, KRT13, KRT79, LARS2, LCK, LECT2, LGALS12, LHX4, LIMA1, LMX1B, LNX1, LOXL1, LRP3, LRP5L, LRRC18, LRRC45, LRRC55, LRRC61, LRRN3, LTBR, LYVE1, MAFB, MAG, MAGT1, MANSC1, MAP1LC3C, MAP2K1, MAP2K4, MAP2K5, MAP6D1, MAPK14, MARVELD2, MCAM, MCF2L, MCM10, MCOLN3, MED1, MEM01, METTL14, MEX3B, MFAP4, MGAT4B, MGME1, MICU2, MIER3, MKS1, MLLT11, MLST8, MMP13, MOBP, MOVIO, MRPL3, MRPS27, MSTN, MTG2, MTHFD2, MTMR12, MTMR8, MYBL1, MYCL, MYO1A, MYORG, NACC2, NADSYN1, NAE1, NBPF15, NCAM1, NCDN, NCLN, NDUFA2, NDUFB7, NECAP1, NECAP2, NECTIN1, NECTIN2, NEK3, NF2, NFIL3, NGFR, NIBAN1, NID2, NIF3L1, NIPSNAP3B, NKX2-8, NOA1, NOXI, NPLOC4, NPNT, NR1I2, NRM, NRTN, NT5DC1, NTPCR, NUP37, NXF3, NXPE3, NXT1, OAT, OIT3, OLFM2, OR1F1, OR1N1, OR52E8, OR9A4, 0RM2, OTUD7B, OTX1, OXCT2, P2RX4, P2RX5, P2RX7, P2RY6, P2RY8, P4HTM, PABPC3, PAF1, PAIP2, PARD6B, PARVA, PAX9, PCCA, PCDHGB1, PCLO, PDE1A, PDE4A, PDE9A, PDGFRA, PDHB, PDSS1, PDZD3, PDZD9, PELO, PFKL, PFKP, PI3, PI4K2A, PIGO, PIM1, PIMREG, PLA2G7, PLAGL1, PLAT, PLEKHO2, PLOD1, PLOD2, PLPP1, POFUT2, POGLUT2, POLR1C, POLR2L, POLR3C, POU3F2, PPA2, PPHLN1, PPP1R16B, PPP1R32, PPP1R9B, PPP2R2B, PPP2R3B, PRAME, PRDX3, PREB, PRELP, PRKCE, PRKCH, PRMT8, PRSS3, PSMB10, PSMC4, PSMD3, PTK6, PTPN18, PTPN2, PTPN9, PYROXD2, R3HDM2, RAB29, RAB30, RAB33B, RAB6B, RAB8B, RAD18, RAET1E, RAG2, RASGRP4, RASSF1, RBM12, RBM46, RCBTB2, RCC1L, RDX, RECQL4, REEP4, REG3A, RET, RGCC, RILP, RIMS2, RIN1, RINL, RITA1, RMND5A, RNF181, RPA3, RPL18, RPL28, RPS6KA2, RRM2, RRP15, RTN2, RUFY4, RUNDC1, SAMD13, SAMD4A, SCEL, SCGB2A2, SDCBP, SDHAF2, SEC23B, SEC63, SEMA3D, SEMA6D, SENP5, SEPTIN6, SEPTIN7, SERPINA10, SERPINE2, SFT2D2, SGCA, SGCB, SGIP1, SH3GL2, SHFL, SHOC2, SIAH2, SIGLEC7, SIRPG, SIRT6, SLC17A4, SLC22A5, SLC23A1, SLC23A3, SLC25A25, SLC25A3, SLC25A40, SLC25A41, SLC27A2, SLC27A3, SLC29A4, SLC2A8, SLC37A2, SLC39A12, SLC41A3, SLC44A5, SLC46A3, SLC51B, SLC6A5, SLCO4A1, SMAD3, SMG9, SMIM19, SMOX, SMPDL3B, SNF8, SNX5, SOCS6, SOD3, SOHLH2, SOX6, SOX8, SOX9, SPAG5, SPATA4, SPATCI, SPNS1, SPOCK1, SPRED1, SPSB1, SRF, SRM, SRPK2, SRRD, SSH3, SSRP1, ST6GALNAC6, ST7, ST8SIA3, STAT4, STIMATE, STING1, STOML2, STX1B, STX5, STXBP2, SYCE1, SYT11, SYT9, TAB2, TAMM41, TBC1D21, TBCD, TBL3, TBRG4, TBX10, TBX21, TBX22, TCF7L2, TERF2IP, TES, TEX10, TEX2, TEX45, TFAP2A, TFF2, TGFB3, THOC1, THOC3, THOP1, THRB, THRSP, THUMPD3, TIPRL, TLE1, TLE4, TMED3, TMEM107, TMEM183A, TMEM64, TMEM98, TMLHE, TMOD4, TNFSF15, TNNT2, TOMM22, TPRG1L, TPST2, TRAF3IP1, TRIM40, TRIM47, TRIP13, TRMT13, TRMT1L, TRMT2A, TRPV2, TRPV5, TSGA10, TSPAN16, TSPAN2, TSPAN32, TSPAN4, TSPAN5, TUBAL3, TUBB, TUBB3, TUBB4A, TUBGCP2, TULP4, TUT7, TXNIP, U2AF1L4, UBA6, UBASH3B, UBE2B, UBE2G1, UBE2I, UBE2O, UBQLN2, UBR2, UGDH, ULK3, UMOD, UNC13D, UQCRC2, UQCRFS1, USP1O, USP28, USP33, UVRAG, VAX2, VEGFA, VNN1, VNN3, VPS72, VRTN, VSIG8, VTI1B, VWC2, WBP11, WDFY2, WDR4, WDR60, WDTC1, WEE1, WFDC13, WFDC6, WNT2B, WRAP73, WSB1, XKR6, XPO7, XRCC5, XXYLT1, YAP1, YBX3, YY1AP1, ZBTB18, ZBTB39, ZDHHC20, ZDHHC5, ZIK1, ZKSCAN1, ZNF101, ZNF16, ZNF175, ZNF224, ZNF25, ZNF334, ZNF350, ZNF462, ZNF485, ZNF519, ZNF653, ZNF677, ZNF71, ZNF761, ZNF776, ZNF785, ZNHIT2, and ZSCAN18.
Embodiment 68. The method of Embodiment 66 or Embodiment 67, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR.
Embodiment 69. The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment.
Embodiment 70. The method of Embodiment 69, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, AL0X15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, AP0A5, AP0C4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, Clorf35, Clorf87, C1QB, C1QL3, C1QTNF12, C3AR1, C7orf26, CACNG3, CACNG6, CALHM1, CALHM3, CAPN2, CAPZA2, CARD8, CASD1, CASK, CATSPER2, CAVIN3, CCDC106, CCDC121, CCDC153, CCDC68, CCDC70, CCDC93, CCL28, CCNL1, CCNY, CCT7, CD300C, CD300LD, CD96, CDC37, CDC42EP2, CDCA2, CDH13, CDH26, CDHR1, CDPF1, CDRT4, CEP120, CEP43, CERKL, CERS2, CES2, CFAP161, CFAP46, CGGBP1, CHID1, CHKA, CHRND, CIART, CITED2, CITED4, CLDN18, CLEC2D, CLSTN1, CLSTN3, CNDP2, COL6A2, C0LCA2, COMT, COPZ2, CORO6, CPA6, CPT1C, CREB3L2, CRLF3, CRP, CSH1, CYP2C8, CYP2D6, CYP51A1, DBT, DCDC2, DCP2, DCUN1D5, DDB1, DDI2, DDX1, DDX53, DEAF1, DELEI, DEXI, DGKG, DHRS13, DKK4, DNAJB8, DNAJC11, DNTT, DOK5, DPP4, DPYSL4, DPYSL5, DSCC1, DUSP8, DYDC2, E2F2, EBP, ECU, EDN3, EGLN3, EIF1AY, EIF2AK1, ELAVL1, ELN, ELOA, ELOA2, EMC3, EMC4, ENKUR, ENOXI, EPB41L1, EPB41L4A, EPHX4, ERICH1, ESRP1, ETFDH, FAM161B, FAM207A, FAM20C, FAM76B, FAM78A, FBXL3, FBXO31, FBXO4, FCGR2A, FCN2, FCRL5, FCRLB, FGF18, FGFR1OP2, FGL1, FHL5, FIGNL1, FLU, FNDC9, FOXA3, FPGS, FREM1, FRMD8, FSD1L, FUZ, FXR1, GABBR2, GABRQ, GATAD1, GDF10, GDF5, GDF6, GGA1, GGT5, GID8, GJA10, GKAP1, GMCL1, GNA12, GNAI3, GNB5, GNL1, GNLY, GORASP1, GORASP2, GPC5, GPN2, GPR15, GPRASP2, GRIK2, GRINA, GRP, GSPT2, GTF2F1, GTPBP2, GXYLT1, H3C10, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HIC2, HINT1, HLA-DOB, HLA- DQB2, HOXB9, HOXD3, HTR1A, HYLS1, IDS, IFNA10, IFNL3, IGFBP1, IGFBP4, IGHG1, IGHV7-81, IK, IL13RA1, IL17C, IL1R2, IL2RG, IMPA1, INKA2, INO80E, INPP5J, IQCG, IQUB, ITGB7, ITLN1, ITPA, JADE1, JAKMIP1, JAML, KCNAB2, KCND1, KCNMB1, KCTD10, KCTD13, KHDRBS3, KIAA0895, KIFC2, KLC2, KLHDC1, KLHL9, KLK10, KPTN, KRT79, KRTAP10-7, KRTAP4-4, L3MBTL4, LAMB3, LCN9, LDHA, LHX2, LIAS, LIMA1, LIMD1, LMNA, LMO2, LOXHD1, LPCAT2, LRFN5, LRP11, LRP3, LRRC18, LRRC45, LRRFIP2, LRTM1, LYPD1, LYPD5, LYPD6B, MACROD1, MACROH2A1, MAFF, MAGEB6, MAGEH1, MALT1, MANF, MAP6D1, MAPK15, MAPKAPK5, MAPRE3, MARCKS, MARS1, MAST2, MAST3, MAST4, MC3R, MCAM, MCM8, MCM9, MCU, MDGA2, METTL25, MGAT4B, MICU2, MIDN, MINDY1, MIPEP, MLST8, MMD, MMP11, MMUT, M0RN1, MOXD1, MPND, MPP2, MPV17L2, MPZL1, MRAP2, MRM3, MRPL3, MRPL4, MRPL41, MRPL48, MRPL51, MRPS30, MRS2, MSMP, MSX1, MSX2, MTMR1, MTPAP, MUC3A, MVB12A, MX1, MXRA8, MYBL1, MYO1A, MYOC, MY0M3, NABP2, NAF1, NAGLU, NAIF1, NAXD, NCCRP1, NCDN, NDOR1, NDUFA7, NDUFS8, NELLI, NEUROG3, NFE2L3, NFIA, NFS1, NHLRC3, NINJ2, NIPAL3, NKAP, NMD3, NONO, NPDC1, NPNT, NR5A1, NRSN2, NTN5, NUBP1, NUDT3, NUDT9, NUP54, NUP62, NUP85, NXNL2, NXPE3, NYX, OAS1, ODF3, ODF3L2, OGGI, OLIG1, OLIG3, OPCML, OR10H1, OR4A15, OR4K17, OR51E1, OR5D14, OR8B12, ORC3, OSGIN1, OSTN, OTUB2, OTUD5, OXGR1, P2RY12, PACC1, PACSIN1, PACSIN2, PAK4, PARG, PARM1, PAX9, PCDH8, PCDHA10, PCDHB13, PCDHB2, PCSK7, PCYOX1L, PDE4A, PDE9A, PDHB, PDIA3, PDZD7, PEMT, PENK, PEX16, PFDN5, PFKFB3, PFKFB4, PFKL, PGRMC1, PHACTR3, PHF7, PICALM, PIGH, PIGP, PKM, PKP1, PLA2G3, PLAT, PLCG2, PLD6, PLEKHG5, PLEKHO2, PLP2, PLPPR2, PLXDC2, PMFBP1, PNMA2, PNOC, PNPT1, PGDN, POGLUT3, POLR3E, POLR3F, POU3F2, PPCDC, PPP1R12C, PPP2R1A, PPP2R2D, PPP3R2, PRDM1, PRKCD, PRLHR, PRMT2, PRNP, PRPF4, PRPF40A, PRR18, PRSS22, PSMA2, PSMD5, PTDSS1, PTGES2, PTK6, PTPN18, PTPRH, PTPRJ, PTPRS, PUM3, PWP1, PXMP4, PYROXD2, QPRT, RAB23, RAB39A, RAB42, RAB6B, RAD51B, RAFI, RALGPS1, RASSF2, RBM24, RBM3, RBM34, RBM46, RCC1L, RCN3, RDH10, RHBDD1, RHOH, RHOXF1, RILPL2, RIN1, RING1, RINL, RITA1, RNF126, RNF141, RNF144B, RNF220, RNF6, RO60, ROR1, RP2, RPH3AL, RPL30, RPL34, RPL6, RRS1, RSAD2, RTL8A, RUNDC1, RUNDC3A, RUSC1, RXFP3, SAFB2, SASS6, SBK1, SCG3, SCNN1A, SDF2L1, SDHAF2, SEC14L2, SEC61G, SEC63, SEL1L2, SEMA3G, SENP5, SERBP1, SERPINB5, SF1, SFRP1, SFT2D2, SFXN3, SGK1, SGO1, SH3GL2, SH3GLB1, SHH, SHKBP1, SHOC2, SIVA1, SKIL, SLC12A1, SLC1A7, SLC22A13, SLC22A7, SLC25A20, SLC25A43, SLC27A6, SLC2A13, SLC2A8, SLC37A3, SLC45A2, SLC49A4, SLF1, SMAD6, SMARCE1, SMG5, SNAP91, SNAPC2, SNRPB, SNRPG, SNX14, SNX27, SOWAHA, SPAG5, SPATS2, SPIRE1, SPRTN, SPTLC2, SRC, SRF, SSH3, SSX3, ST3GAL6, STARD7, STIM1, STK3, STK35, STMN1, STOML3, STX10, STXBP4, SUSD3, SUSD6, SYP, TAC1, TAFA5, TBC1D19, TBCK, TBL3, TBRG4, TCAF1, TCEA2, TCF19, TCP11, TCP11L1, TDG, TDP2, TEX13A, TGM4, TGOLN2, THAP11, THBD, THOC5, TIMM29, TLR2, TM4SF4, TMED6, TMEM106B, TMEM178B, TMEM204, TMEM234, TMEM263, TMEM41A, TMEM86A, TMIGD2, TMPRSS1 IE, TMPRSS2, TNFAIP1, TNFAIP8L1, TNFRSF10C, TNFSF13B, TOR1B, TOX2, TPD52L2, TPM4, TPO, TPSD1, TPT1, TRAPPC10, TRAPPC8, TRIM40, TRIM55, TRIR, TRMT12, TRPC5, TRPV2, TSKS, TTC12, TTLL7, TUBA1C, TUBGCP2, TXNDC5, UBAC1, UBXN2A, UGT3A1, UIMC1, UNCI 19, UQCRFS1, USH1C, USP15, USP21, VAC14, VEGFA, VPS37B, VPS45, VRTN, WAPL, WDR1, WDR24, WDR54, WDR5B, WDR60, WDR61, WIPI2, WNT2, XAF1, YBX2, ZAP70, ZBTB5, ZBTB46, ZC2HC1A, ZC3H3, ZCCHC8, ZDHHC1, ZDHHC13, ZFP2, ZFP36L2, ZGRF1, ZMPSTE24, ZNF175, ZNF19, ZNF205, ZNF274, ZNF428, ZNF436, ZNF502, ZNF558, ZNF624, ZNF668, ZNF71, ZNF710, ZNHIT2, and ZSWIM1.
Embodiment 71. The method of Embodiment 69 or Embodiment 70, wherein the immunosuppressive resistance gene is ZBTB46.
Embodiment 72. The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
Embodiment 73. The method of Embodiment 72, wherein the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD 10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, AD ATI, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1, B9D2, BAB AMI, BAG5, BCHE, BLK, BMPR1B, BPIFC, BRF2, BSND, BTNL3, BYSL, C10orf82, C18orf25, C18orf54, Clorfll5, Clorf43, Clorf56, C1QTNF2, C1R, C2CD2, C5orfl5, C6orfl20, CAB, CA5B, CA8, CA9, CABS1, CALCOCO1, CALCR, CALHM3, CAMK2A, CAMLG, CARD8, CASP1, CASP7, CASTOR1, CBX7, CCDC110, CCDC69, CCDC82, CCDC84, CCL2, CCL21, CCR8, CCSER1, CD151, CD300LF, CD48, CD83, CD86, CDH7, CDK1, CDPF1, CDR2, CELF1, CEP63, CERS1, CES3, CFAP410, CGGBP1, CHAF1B, CHMP2A, CHMP7, CHRM1, CHST9, CISH, CLC, CLDN6, CLEC5A, CLIC5, CLP1, CLTRN, CLUAP1, CMTM7, CNTF, COG3, COL25A1, COL8A2, COMMD4, COPZ1, COPZ2, COQ4, COX6B2, CPLX2, CRACR2B, CROT, CRTAC1, CRY2, CSF1, CSNK1G2, CST9, CSTF3, CTDP1, CTNNA2, CTSF, CXCR6, CXXC1, CYP27A1, CYP2D6, CYTL1, DAXX, DBN1, DDHD2, DDX24, DES, DEXI, DGLUCY, DHX36, DNAI2, DNAJA2, DNAJB5, DNAJB6, DNAJC11, DNAJC27, DNAJC6, D0K5, D0K6, DRGX, DUS1L, DUSP5, DZIP1L, ECI2, EFCAB7, EGFL6, EGLN3, EIF3K, EIF4EBP1, ELSPBP1, EMC3, EPDR1, EPN1, EPO, ERFE, ERVK3-1, ESMI, F2RL2, FAAP100, FADS1, FAM172A, FAM53C, FAM71C, FAM81A, FBLN5, FBXL16, FBXO7, FBXW11, FCGR3A, FCRL5, FDFT1, FETUB, FEZF2, FGF10, FGF19, FGL1, FKBP5, FKBP9, FMOD, FNDC9, FOSB, FRMD3, FRMD5, FRZB, FUT3, GAB3, GABRA4, GABRG2, GALNT2, GALNTL6, GAS7, GBA2, GBP6, GEMIN8, GET1, GFM2, GFRA3, GK2, GLIPR1, GLRA2, GLRB, GLYCTK, GNA14, GNB3, GNL3, GOLM2, GPATCH2L, GPATCH3, GPM6B, GPR176, GPR45, GRAMD1B, GRB7, GRK2, GRK7, GSTM3, GXYLT1, GZMM, HAPLN3, HAUS2, HBG2, HCRTR1, HDAC8, HDGF, HEMK1, HERPUD1, HESX1, HEXA, HMHB1, HOXB5, HOXB6, HOXD3, HOXD4, HPGDS, HSD17B6, HSD17B8, HSPA2, HTN1, HTR5A, IFITM3, IFNA10, IGF1, IGFALS, IGHM, IL11RA, IL17A, IL17RE, IL2RB, IL12RB2, IL4, INSL6, ISL2, ISM2, IST1, ITPRID2, JAML, JUN, JUNB, KBTBD7, KCNA6, KCNN3, KEAP1, KIF3A, KIFC2, KIR2DL1, KLHDC2, KLHDC7B, KLHL9, KRT19, KRT6A, LARS2, LCK, LCN1, LDAH, LDLRAD4, LETMD1, LHX4, LHX9, LIN28A, LIPG, LNX1, LPAR5, LPL, LRFN3, LRRC15, LRRC2, LRRC34, LRRC42, LTBR, LYZ, MAF1, MAGOH, MAN1A1, MAN1B1, MAP2K6, MAP3K7, MAP4K5, MAPKAPK2, MAPKAPK5, MARCHF1, MARCHF2, MBNL1, MBP, MC3R, MCAM, MED26, MEIS3, METTL27, METTL2B, MIA2, MIF, MIPOL1, MLKL, MMD, MMP10, MOB4, MPHOSPH8, MPND, MPZL1, MR1, MRAS, MRM3, MRPL21, MRPS24, MRPS28, MS4A3, MS4A5, MSRA, MTA2, MTA3, MTHFD2, MTMR3, MTPAP, MTRF1L, MUS81, MYCL, MYCN, MYL10, MYL9, MYOC, MY0M3, MYORG, NAA80, NAPSA, NARF, NAT1, NCK1, NDE1, NDOR1, NDUFA13, NDUFA4, NEK5, NELFE, NFIB, NFKBIB, NINJ2, NIPAL1, NKAIN2, NKAP, NMB, NOC4L, NPIPB15, NRARP, NRSN2, NTF3, NXNL2, OBP2A, OLR1, OR10AG1, OR10K2, OR14C36, OR1F1, OR2M3, OR2T8, OR5C1, OR7A5, OR9Q1, ORAI3, ORC2, ORM1, ORM2, OSGIN1, OSR2, OTUD5, P3H4, P4HA3, P4HTM, PAK2, PAK4, PBX2, PCDHA2, PCDHB12, PCDHGA2, PDYN, PFDN4, PFKP, PFN4, PGK1, PGLYRP1, PHF23, PHF7, PHKG1, PHOSPHO1, PI15, PI3, PI4KB, PITHD1, PKIA, PKNOX2, PLA2G3, PLA2G7, PLAUR, PLEKHA8, PLEKHO2, PLET1, PLOD2, PNPT1, POPDC3, PPM1D, PPME1, PPP1R2, PPP1R32, PPP2R2B, PPP2R3C, PPP2R5C, PPP6R2, PRKCB, PRKRIP1, PRKY, PRMT8, PRSS3, PRUNE2, PSCA, PSG1, PTGER3, PTK6, PT0V1, PTPRO, PTTG1IP, PUDP, PWWP3B, PYCARD, PYGB, QPCT, QPRT, RABI IB, RAB25, RAB28, RAB34, RAB40B, RABEP2, RADU, RAET1E, RAMP1, RARS1, RAX2, RBBP5, RCHY1, RCN3, REEP2, RFC4, RFPL2, RFX3, RIBCI, RINL, RIPK4, RLBP1, RNASE9, RNASET2, RNF111, RNF112, RNF144B, RNF24, RNF38, RNF7, ROPN1L, RP9, RPL6, RPS2, RPS3A, RPS6, RRAGA, RRAGD, RRP1, RRP9, RSAD1, RUBCN, RUNX1T1, RUVBL1, SAMHD1, SARS1, SCNN1A, SCNN1B, SCNN1G, SEL1L2, SELENBP1, SEPHS1, SEPTIN10, SEPTIN12, SERINC2, SERPINA3, SERPIND1, SERPINE1, SERPINE3, SETD3, SFRP4, SGK2, SH3KBP1, SHARPIN, SHISA3, SHOC2, SHOX, SIGLEC7, SIRPB2, SIRPG, SIRT3, SKIL, SLC13A1, SLC14A1, SLC20A2, SLC22A13, SLC22A31, SLC22A8, SLC25A1, SLC25A46, SLC25A47, SLC25A48, SLC2A8, SLC37A3, SLC39A7, SLC5A12, SLC6A19, SLC6A7, SLC7A9, SLCO1A2, SLFNL1, SMG9, SMPX, SNORC, SNRNP25, SNRPB, SNRPN, SNX16, SOAT1, SOCS5, SPATA22, SPATS2, SPC25, SPG21, SPINT1, SPINT2, SPNS2, SPP2, SQOR, SRC, SRFBP1, SRP54, SRP9, SRSF9, SSBP2, SSPN, STAP1, STARD7, STIM1, STK11, STX8, SULT4A1, SUMF2, SURF6, SYMPK, SYNCRIP, SZT2, TAS2R40, TAS2R60, TBCC, TBRG4, TBX20, TBX3, TON, TCEA1, TCEA2, TCF19, TCF7L2, TCN2, TCTN1, TDP2, TENT5C, TEX35, TFCP2L1, TFDP2, TGFB3, THAP12, TIAM2, TICAM2, TIPIN, TKT, TLE4, TM2D2, TM9SF3, TMEM106B, TMEM143, TMEM160, TMEM211, TMEM237, TMEM270, TMEM30A, TMEM39B, TMEM45A, TMEM68, TMPRSS3, TNFSF12, TOMM70, TOR1AIP1, TOX, TPP1, TPRKB, TPST2, TRAPPC12, TRDMT1, TRIM10, TRIM47, TRIM63, TRIP10, TRMO, TRMT44, TRPV4, TSN, TSPAN31, TSPEAR, TSSK3, TTBK2, TTC32, TUBA3C, TUBA3D, TUT7, TYW3, UBA1, UBASH3B, UBE2S, UBXN2A, UCHL3, UCHL5, UQCR10, USP1, USP19, VAT1L, VMA21, VPS29, VPS36, VSTM2A, VWA1, WAS, WDFY1, WDR4, WDR59, WDR63, WDR78, WNT11, WNT3A, WNT9A, YAF2, YJU2, ZBTB48, ZC3HAV1L, ZCCHC2, ZCCHC7, ZFP36L2, ZIC3, ZNF165, ZNF830, ZP2, ZSCAN21, and ZSCAN9.
Embodiment 74. The method of Embodiment 72 or Embodiment 73, wherein the immunosuppressive resistance gene is selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL.
Embodiment 75. The method of Embodiment 64, wherein the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment. Embodiment 76. The method of Embodiment 75, wherein the immunosuppressive resistance gene is selected from the group consisting of AB AT, AB HD 12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, AL0XE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARE4D, ARMC2, ARNTE, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GAENT2, B3GAET4, B3GNT2, BAB AMI, BAP1, BCAP31, BCCIP, BCE2E2, BCR, BDNF, BECN1, BEND2, BEND7, BEOC1S4, BMP5, BMPR1B, BRD3, BRF2, BRINP3, BTBD17, BTNE3, C10orf62, C12orf42, C14orf28, Clorf210, C2orf78, C6, C7orf31, CAB, CACNB3, CAER3, CARD14, CARD19, CASC1, CASP10, CASQ2, CBFB, CBX3, CBX4, CCDC141, CCDC148, CCDC68, CCDC8, CCDC82, CCIN, CCNG2, CCNY, CCR1, CCR10, CCR3, CD19, CD244, CD47, CD5E, CD83, CDC42EP1, CDC42EP4, CDCA2, CDCP2, CDH13, CDYE2, CEP120, CEP63, CERCAM, CERKL, CFAP100, CFAP20, CFAP410, CFAP47, CFAP91, CFHR1, CHGA, CHUK, CIRBP, CLCNKB, CLDND1, CLU, CMTR1, CNPPD1, COA3, COG1, COL22A1, COL25A1, COL6A2, COL8A2, COPB1, COPS3, COQ3, COQ8B, COX18, CPOX, CPQ, CPT1A, CPXM1, CRACR2A, CREB3L1, CRNN, CRTAP, CRYGD, CS, CSF1, CSF1R, CSF2RB, CSF3R, CSGALNACT1, CSTF2, CTCF, CTSV, CUEDC1, CUL2, CXADR, CYB5R2, CYP20A1, CYP26A1, CYP2C19, CYP8B1, DAAM2, DAB1, DACT2, DARS1, DBN1, DCAF4L2, DCAF8, DCT, DDO, DDR1, DDX43, DDX54, DEAF1, DECR1, DENR, DESI2, DHX40, DIP2A, DKK4, DNAAF3, DNAI2, DNAJC7, DNAL1, DPP4, DPT, DPYSL4, DRG1, DSCAM, DTL, DTX2, DUS1L, DUSP12, E2F7, ECHI, EFEMP2, EFHC1, EFS, EIF2B3, EIF4A2, EIF5, ELAVL4, ELL, ELMOD3, ELN, ELOA, ELOA2, ELP3, EMC7, ENCI, ENDOV, ENPP3, ENTPD5, EOLA2, EPB41L1, EPHB6, EPN1, ERCC6, ESRP1, ESRRA, ETFDH, EXO1, EXOC3, EXOSCIO, EXOSC8, EYS, F3, FABP6, FAM117A, FAM117B, FAM118B, FAM161B, FAM200A, FAM20A, FAM47A, FARSA, FBLN1, FBLN5, FBP1, FBXO24, FBXO40, FCHO1, FCRL5, FCRLB, FECH, FEM1B, FGF3, FGG, FKBP6, FLVCR1, FMNL1, FM05, FNIP1, FOS, FOSB, FOXD4, FOXJ1, F0XM1, FOXN3, FOXRED2, FREM1, FRMD5, FRS2, FSTL1, FSTL4, FSTL5, FTMT, FXR2, G6PD, GABPB1, GABRB1, GALNT7, GANAB, GAPDHS, GATA2, GBP4, GCAT, GCGR, GCNT7, GCSAML, GDF2, GDF6, GET4, GGA1, GHDC, GINS4, GK, GKAP1, GLB1, GLRA2, GLRX5, GLYR1, GMCL2, GNA14, GNAT2, GNB3, GNL1, GNL3, GPAT4, GPC3, GPC4, GPC5, GPR84, GRHL3, GRHPR, GRIA4, GRID1, GRIK3, GRK4, GRM3, GRM8, GRN, GSDME, GSK3A, GSN, GTF2A1, GTF2B, GTF2E1, GUCY1B1, GYSI, HABP2, HADHA, HADHB, HDAC10, HEPHL1, HERPUD2, HES1, HEXD, HHIPL2, HINT2, HLA-C, HOMER3, HOOK3, HOXB6, HOXD3, HPS3, HPS5, HSD11B2, HSD17B13, HSD17B7, HSD3B1, HSPB9, HTR2B, IFNA6, IFNL1, IFT27, IGHA1, IGSF1O, IGSF21, IL1ORB, IL12RB2, IL13RA1, IL21, IL26, IL7R, ILVBL, IMPG1, INA, INHA, INSL4, INTU, IQUB, IRAG1, IRAKI, IREB2, IRF3, IRF5, IRX3, ITFG1, ITGB2, ITGB7, ITIH5, ITM2B, JAKMIP1, KANSL3, KAT5, KCNG3, KCNJ14, KCNK12, KCNK2, KCNK9, KCNMB3, KCTD12, KCTD4, KIAA2013, KIF2C, KIF3A, KIF3B, KIFC2, KITLG, KLC3, KLHDC2, KLHL13, KLHL21, KLHL8, KLK1, KRT6A, KXD1, L3MBTL4, LAD1, LAMP1, LAP3, LARS2, LDLRAD3, LGMN, LHX2, LHX4, LIMA1, LIMD1, LIPH, LIPT1, LKAAEAR1, LNPEP, LOXHD1, LOXL3, LTA4H, LTBR, LUC7L2, LYL1, LZTS2, MAG, MAN1A1, MANEA, MANF, MAP3K7, MAP4K5, MAP6D1, MAPK15, MAPK8, MAPKAPK5, MAPKBP1, MARS2, MATN2, MBLAC1, MCAM, MCOLN2, MCOLN3, MDH1B, MDM4, MEAK7, MECP2, MED1, MEOX1, METAP1, MFAP3L, MFNG, MITD1, MLEC, MLH1, MLLT3, MMP10, MMP16, MMS19, MNAT1, MON1B, MPP2, MPP7, MRM3, MRPL19, MRPL3, MRPL47, MSLN, MSRA, MTARC2, MTHFR, MTMR4, MTMR6, MUTYH, MVP, MYBL1, MYCN, MYO1A, MYO5C, MYOC, MYOM3, MYORG, MYT1, NAGLU, NAMPT, NCKIPSD, NDC80, NDRG4, NDUFAF7, NDUFB6, NDUFS8, NDUFV2, NECTIN4, NELFA, NFIB, NIF3L1, NIPAL1, NIPBL, NMD3, NOC2L, NPLOC4, NPY, NPY2R, NQO1, NR2E1, NR6A1, NT5DC2, NTM, NTN5, NTNG1, NUDT8, NUP210, NXPE3, OAS1, OAS2, ODF2, OGA, OGFRL1, OLFM2, OLFML1, OLFML2B, OLIG1, OLR1, OMP, OR4D1, OR4S2, OR52N5, OR52W1, OR5B3, OR9Q1, ORMDL2, OSBPLIO, OSGEPL1, OTX1, OXSR1, P2RX6, P2RY1, PACS2, PAEP, PAFAH1B2, PAK4, PARL, PARP9, PARS2, PC, PCDHA1, PCDHA6, PCDHGA2, PCDHGA5, PCSK2, PDCD6, PDE4A, PDIA3, PDK2, PDZD9, PENK, PFKL, PGAM5, PGK1, PGM2, PHF11, PHYH, PICALM, PIK3R1, PIP5K1B, PLAGL1, PLCD4, PLD1, PLEK, PLEKHG5, PLEKHS1, PLIN1, PLK1, PLPPR2, PM20D1, PNMA8A, POLE, POLI, POLR2K, POLR3F, POU3F2, PPA2, PPIG, PPM1D, PPP1R12C, PPP1R16B, PPP1R32, PPP2R3C, PPP2R5C, PPP2R5D, PRAC1, PRAM1, PRAME, PRDM1, PRDX3, PRKAA2, PRKAG2, PRKAR1B, PRKCE, PRMT1, PROC, PRRT2, PRSS45P, PRSS48, PSAPL1, PSD3, PSEN1, PSMC4, PSMD14, PTBP3, PTCD2, PTDSS1, PTGER2, PTK6, PTPN12, PTPRN, PUF60, PUM1, PYDC1, PYGB, RAB1A, RAB33B, RAB42, RABL6, RAD18, RAD51, RAET1G, RALBP1, RASSF4, RBBP7, RBM12, RBM38, RBM4, RBM46, RBM4B, RBX1, RCBTB2, RCC1L, RDH10, RDH12, REC8, REN, RET, RFC2, RFC5, RFX4, RGS16, RHAG, RHEX, RH0BTB2, RICTOR, RIMBP2, RIMS3, RIOK3, RIPK1, RIPK2, RIT1, RMDN3, RNASEH2B, RNF114, RNF148, RNF213, RNF6, RNPEPL1, RO60, RORA, RPL30, RPS14, RPS4X, RPS6KA2, RPS6KB1, RSRC1, RTF1, RTN1, RUBCN, RUNDC1, RUNX3, S100PBP, SAMHD1, SCAMP2, SCIN, SCNN1D, SCRN2, SDSL, SEC63, SEL1L3, SELL, SENP3, SEPTIN10, SEPTIN8, SERINC3, SERPINF1, SGK3, SGMS1, SH3GLB1, SHOC2, SIAH2, SIGLEC10, SIGLEC12, SIGLEC7, SIRPG, SIRT5, SKAP1, SKIL, SLC15A3, SLC16A1, SLC16A7, SLC18A2, SLC1A7, SLC20A2, SLC22A2, SLC22A23, SLC22A24, SLC23A2, SLC25A19, SLC25A25, SLC25A47, SLC26A2, SLC2A4, SLC2A8, SLC36A3, SLC37A2, SLC37A3, SLC39A14, SLC43A1, SLC5A11, SLC5A12, SLC5A7, SLC6A7, SLC7A1, SLCO1A2, SLITRK3, SMARCD3, SMOX, SNCAIP, SNTA1, S0CS6, SOX9, SPATA2, SPCS3, SPN, SPNS2, SPOCK3, SP0UT1, SPRR4, SRC, SRD5A3, SRFBP1, SRMS, SSMEM1, SSX2IP, ST7L, STEAP1, STIM1, STK11, STK17A, STK17B, STK3, STK39, STXBP1, STXBP2, STXBP3, STXBP5, SUGCT, SUN5, SUSD2, SYNCRIP, TAB2, TBC1D10A, TBC1D22B, TBC1D9B, TBCD, TBL1X, TBX6, TC2N, TCF25, TCTN2, TDGF1, TEAD2, TESK1, TESK2, TEX48, TFAP2A, TFAP4, TGFB1, TGFBI, TGOLN2, THAP3, THAP7, THEM4, THOC1, THOC7, THOP1, THSD4, TIAM2, TICAM2, TLE6, TLK1, TM9SF4, TMED2, TMEM259, TMEM62, TMIE, TMLHE, TMOD1, TMTC3, TNF, TNS1, TOMM70, TRAF3IP1, TRAM1, TRAP1, TRAPPC3, TRAPPC4, TRAPPC8, TRAPPC9, TRIB3, TRIM32, TRMT13, TRPM3, TRPV4, TSGA10, TSPEAR, TSPYL6, TTC13, TTC16, TTC26, TTC38, TTC8, TTF2, TTLL2, TTLL7, TUBA3E, TUBA8, TUBB3, TUBB4B, TUFM, TYMS, UBE2O, UBE2Z, UBE3A, UBE3C, UBOX5, UGDH, UGP2, UGT3A1, UGT8, UMOD, USP1, USP49, UXS1, VANGL2, VASN, VEGFA, VIL1, VPS45, VWA1, WAPL, WDR37, WDR63, WDR90, WDR91, WNT4, WWOX, XRCC5, YBX2, YME1L1, ZBTB14, ZBTB18, ZBTB46, ZC3H10, ZC3H11A, ZCCHC8, ZDHHC11, ZDHHC15, ZDHHC6, ZFYVE21, ZGRF1, ZMAT3, ZNF165, ZNF18, ZNF19, ZNF20, ZNF224, ZNF25, ZNF277, ZNF334, ZNF350, ZNF354C, ZNF396, ZNF398, ZNF436, ZNF461, ZNF467, ZNF496, ZNF560, ZNF565, ZNF566, ZNF597, ZNF610, ZNF623, ZNF668, ZNF74, ZP1, ZPLD1, ZSCAN25, and ZSWIM2. Embodiment 77. The method of Embodiment 75 or Embodiment 76, wherein the immunosuppressive resistance gene is selected from the group consisting of CD47,
CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZTBTB46.
Embodiment 78. A method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of:
(i) collecting peripheral blood mononuclear cells (PBMCs) from an individual,
(ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i),
(iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and
(iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
Embodiment 79. The method of Embodiment 78, wherein the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR).
Embodiment 80. The method of Embodiment 78 or Embodiment 79, wherein the exogenous nucleic acid further comprises a T cell receptor (TCR).
Embodiment 81. The method of any one of Embodiments 78-80, wherein the peripheral blood mononuclear cells are obtained from leukapheresis.
Embodiment 82. The method of any one of Embodiments 78-81, wherein the CD8+ and CD4+ T cells are isolated sequentially.
Embodiment 83. The method of any one of Embodiments 78-82, wherein the naive CD4+ T cells are differentiated into activated CD4+ T cells.
Embodiment 84. The method of any one of Embodiments 78-83, wherein the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
Embodiment 85. The method of any one of Embodiments 78-84, wherein the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
Embodiment 86. The method of any one of Embodiments 78-85, wherein the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
Embodiment 87. The method of any one of Embodiments 78-86, wherein the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection. Embodiment 88. The method of any one of Embodiments 78-87, wherein the transduced lymphocytes are enriched by positive selection.
Embodiment 89. The method of Embodiment 88, wherein the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
Embodiment 90. A method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising:
(i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual,
(ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes,
(iii) transiently stimulating the transduced lymphocytes,
(iv) exposing the transduced lymphocytes to an immunosuppressive environment,
(iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and
(v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene.
Embodiment 91. The method of Embodiment 90, wherein the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
Embodiment la. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
Embodiment 2a. The modified lymphocyte of Embodiment la, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene. Embodiment 3a. The modified lymphocyte of Embodiment la or Embodiment 2a, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 4a. The modified lymphocyte of any one of Embodiments la-3a, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FOSB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
Embodiment 5a. The modified lymphocyte of any one of Embodiments la-3a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, ADARB1, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, D0K5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EX01, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FM05, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, L0XHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MC0LN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, ND0R1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
Embodiment 6a. The modified lymphocyte of any one of Embodiments la-5a, wherein increased expression of the immunosuppressive resistance gene results in increased proliferation and/or increased effector function of the modified lymphocyte in in vitro or in vivo cell proliferation assays mimicking a tumour microenvironment compared to an unmodified lymphocyte.
Embodiment 7a. The modified lymphocyte of any one of Embodiments la-6a, wherein the exogenous nucleic acid further encodes at least two immunosuppressive resistance genes set forth in Table 6.
Embodiment 8a. The modified lymphocyte of any one of Embodiments la-7a, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Embodiment 9a. The modified lymphocyte of Embodiment 8a, wherein the lymphocyte further comprises an expression cassette comprising the nucleic acid encoding the CAR or TCR.
Embodiment 10a. The modified lymphocyte of Embodiment 8a or Embodiment 9a, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in the same expression cassette.
Embodiment I la. The modified lymphocyte of Embodiment 8a or Embodiment 9a, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in separate expression cassettes.
Embodiment 12a. The modified lymphocyte of any one of Embodiments la-1 la, wherein the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
Embodiment 13a. The modified lymphocyte of any one of Embodiments la- 12a, wherein the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs).
Embodiment 14a. The modified lymphocyte of any one of Embodiments la- 13a, wherein the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
Embodiment 15a. The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
Embodiment 16a. The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
Embodiment 17a. The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a TCRy8+ lymphocyte.
Embodiment 18a. The modified lymphocyte of any one of Embodiments la-14a, wherein the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
Embodiment 19a. The modified lymphocyte of any one of Embodiments la- 14a, wherein the modified lymphocyte is a regulatory T cell.
Embodiment 20a. The modified lymphocyte of any one of Embodiments la- 19a, wherein the modified lymphocyte is a human lymphocyte.
Embodiment 21a. The modified lymphocyte of any one of Embodiments la- 20a, wherein the modified lymphocyte is an autologous lymphocyte.
Embodiment 22a. The modified lymphocyte of any one of Embodiments 3a-21a, wherein the modified lymphocyte comprises a vector comprising the expression cassette. Embodiment 23a. The modified lymphocyte of any one of Embodiments 3a- 22a, wherein the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
Embodiment 24a. The modified lymphocyte of Embodiment 23a, wherein the promoter is a ubiquitous promoter.
Embodiment 25a. The modified lymphocyte of Embodiment 24a, wherein the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate- early enhancer and chicken beta-actin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’ LTR, and CMV.
Embodiment 26a. The modified lymphocyte of Embodiment 23a, wherein the promoter is an inducible promoter.
Embodiment 27a. The modified lymphocyte of any one of Embodiments 23a-26a, wherein the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
Embodiment 28a. The modified lymphocyte of any one of Embodiments 23a- 27a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte.
Embodiment 29a. The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene.
Embodiment 30a. The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus.
Embodiment 31a. The modified lymphocyte of any one of Embodiments 23a-28a, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
Embodiment 32a. A vector comprising nucleic acid encoding an immunosuppressive resistance gene set forth in Table 5 or Table 6, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof. Embodiment 33a. The vector of Embodiment 32a, wherein the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 34a. The vector of Embodiment 32a or Embodiment 33a, further comprising nucleic acid encoding a therapeutic protein.
Embodiment 35a. The vector of Embodiment 34a, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
Embodiment 36a. The vector of Embodiment 35a, wherein the nucleic acid encoding the CAR or TCR are included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
Embodiment 37a. The vector of Embodiment 35a, wherein the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or TCR are included in a second expression cassette.
Embodiment 38a. The vector of any one of Embodiments 32a-37a, wherein the vector is a viral vector.
Embodiment 39a. The vector of any one of Embodiments 32a-38a, wherein the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
Embodiment 40a. The vector of Embodiment 39a, the vector is an episomal or nonintegrating vector.
Embodiment 41a. The vector of Embodiment 40a, wherein the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
Embodiment 42a. The vector of any one of Embodiments 32a-37a, wherein the vector is a non-viral vector.
Embodiment 43a. The vector of Embodiment 42a, wherein the non-viral vector is a plasmid.
Embodiment 44a. The vector of any one of Embodiments 32a-43a, wherein the vector further comprises nucleic acid encoding a drug-resistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
Embodiment 45a. A modified lymphocyte comprising one or more of the vectors as defined in any one of Embodiments 32a-44a.
Embodiment 46a. A composition comprising the modified lymphocyte as defined in any one of Embodiments la-3 la. Embodiment 47a. The composition of Embodiment 46a, wherein the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
Embodiment 48a. A method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising increasing expression of an immunosuppressive resistance gene set forth in Tables 5-6 or introducing into the lymphocytes the vector of any one of Embodiments 32a-44a.
Embodiment 49a. The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a tumor microenvironment.
Embodiment 50a. The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
Embodiment 51a. The method of Embodiment 50a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, ADARB1, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP6V1C1, ATP6V1D, ATPAF1, ATRAID, ATRIP, AUTS2, AZI2, B3GNT9, BAIAP2, BAIAP2L2, BAP1, BATE, BCAT1, BCKDHA, BCL2L13, BCL2L2, BLMH, BMPER, BOD1, BPIFA1, BSG, BSPRY, BTC, BTD, BTRC, C18orf54, C1QTNF12, C1QTNF4, C3orf38, C6orf223, C8orf76, C9orfl l6, CALCOCO1, CALHM1, CAT, CAVIN1, CAVIN4, CBWD1, CCDC113, CCDC121, CCDC14, CCDC174, CCDC86, CCDC93, CCIN, CCN2, CCN3, CCR6, CCSAP, CCT7, CD19, CD1B, CD36, CD46, CD86, CD96, CDC14A, CDH1, CDK2AP1, CDK7, CELA2B, CEP97, CFAP46, CFAP47, CHAMP1, CHMP2B, CHST8, CKB, CLEC11A, CLEC4M, CLEC9A, CLPB, CLPSL1, CLSTN1, CLUL1, CLYBL, CNDP2, CNTROB, COA6, COL15A1, COL21A1, COL22A1, COLQ, COMMD5, COPZ2, CORO6, COX11, CPA1, CPSF6, CPT2, CRB3, CREB3L2, CREM, CRHR2, CRTC2, CRYGD, CSK, CTBP2, CTNNA2, CTSS, CX3CR1, CYP20A1, CYP2C9, DADI, DAXX, DBF4, DCDC2, DCLK2, DCTN3, DCX, DDB1, DDHD1, DDO, DDOST, DDX21, DDX56, DEGS2, DEPDC7, DFFB, DIXDC1, DLK1, DNAI2, DNAJC16, DPPA4, DPYSL3, DPYSL4, DRG2, DUT, E2F7, EBNA1BP2, ECHDC1, ECHDC3, ECU, ECRG4, EDC3, EFCAB1, EFCAB12, EFCAB13, EFEMP2, EGR2, EIF2A, EIF4G3, EMP1, ENPP3, EPHA4, EPHB6, EPX, ERCC2, ERN2, ERV3-1, ESRP1, ETNK2, EVI2B, EX01, EXTL3, EZH2, F10, F13B, F2, FAF2, FAM110A, FAM13C, FAM161B, FAM181A, FAM200A, FAM71A, FAM98A, FANCB, FATE1, FBXL13, FBXL16, FBXL20, FCRLB, FEM1C, FEV, FGF22, FGF3, FKBP14, FKBP5, FKBPL, FLOT1, FLVCR1, FLYWCH1, FMN1, FMO5, FOSB, FOXJ1, FOXS1, FPGT, FRMD5, FUT8, G6PC3, GAB3, GABRA5, GABRB2, GABRQ, GAD2, GALNT7, GAN, GAPDHS, GAPVD1, GBP4, GCNA, GDF6, GDF9, GDPD5, GET1, GET4, GGA1, GGA2, GGTLC1, GIMAP8, GKAP1, GLRB, GLT6D1, GLTPD2, GNAI2, GORASP1, GORASP2, GOSR1, GPAA1, GPC5, GPR119, GPR37, GPR65, GPRASP2, GPRC5C, GPT2, GPX1, GRHL2, GRID1, GRINA, GSDME, GTF2H1, GTF2I, GTPBP2, GUSB, GYPA, H2BC4, HABP4, HACD3, HAGH, HAND2, HARS2, HAUS7, HAX1, HDAC9, HDHD3, HENMT1, HEPACAM2, HHEX, HIBCH, HID1, HK1, HLA-C, HLCS, HNF4A, HNRNPAB, HNRNPD, H0XA11, H0XD8, HP1BP3, HPS3, HPS4, HSDL1, HSP90B1, HTR1F, HTRA4, HVCN1, IBSP, IDS, IFNA8, IFNAR2, IFNGR1, IFNLR1, IGFBP1, IGKV1D-17, IHO1, IK, IL10RA, IL12RB2, IL13RA2, IL17RE, IL36G, IMPDH2, INF2, INKAI, INTS5, IP6K2, IPO5, IQCA1, IREB2, JAGN1, JMJD8, KAT14, KAT7, KAZALD1, KCNAB3, KCNG3, KCNJ14, KCNK12, KCNK4, KCNK9, KCNMB2, KCNV1, KCTD4, KHDRBS2, KHDRBS3, KIAA0930, KIF7, KIFC3, KLHL14, KLHL2, KLHL8, KREMEN2, KRT13, KRT79, LARS2, LCK, LECT2, LGALS12, LHX4, LMX1B, LNX1, LOXL1, LRP3, LRP5L, LRRC18, LRRC45, LRRC55, LRRC61, LRRN3, LTBR, LYVE1, MAFB, MAG, MAGT1, MANSC1, MAP1LC3C, MAP2K1, MAP2K4, MAP2K5, MAP6D1, MAPK14, MARVELD2, MCAM, MCF2L, MCM10, MCOLN3, MED1, MEM01, METTL14, MEX3B, MFAP4, MGAT4B, MGME1, MICU2, MIER3, MKS1, MLLT11, MLST8, MMP13, MOBP, MOVIO, MRPL3, MRPS27, MSTN, MTG2, MTHFD2, MTMR12, MTMR8, MYBL1, MYCL, MY01A, MYORG, NACC2, NADSYN1, NAE1, NBPF15, NCAM1, NCDN, NCLN, NDUFA2, NDUFB7, NECAP1, NECAP2, NECTIN1, NECTIN2, NEK3, NF2, NFIL3, NGFR, NIBAN1, NID2, NIF3L1, NIPSNAP3B, NKX2-8, NOA1, NOXI, NPLOC4, NPNT, NR1I2, NRM, NRTN, NT5DC1, NTPCR, NUP37, NXF3, NXPE3, NXT1, OAT, OU3, OLFM2, OR1F1, OR1N1, OR52E8, OR9A4, 0RM2, OTUD7B, OTX1, OXCT2, P2RX4, P2RX5, P2RX7, P2RY6, P2RY8, P4HTM, PABPC3, PAF1, PAIP2, PARD6B, PARVA, PAX9, PCCA, PCDHGB1, PCLO, PDE1A, PDE4A, PDE9A, PDGFRA, PDHB, PDSS1, PDZD3, PDZD9, PELO, PFKL, PFKP, PI3, PI4K2A, PIGO, PIM1, PIMREG, PEA2G7, PEAGE1, PEAT, PLEKHO2, PLOD1, PLOD2, PLPP1, POFUT2, POGLUT2, POLR1C, POLR2L, POLR3C, POU3F2, PPA2, PPHLN1, PPP1R16B, PPP1R32, PPP1R9B, PPP2R2B, PPP2R3B, PRAME, PRDX3, PREB, PRELP, PRKCE, PRKCH, PRMT8, PRSS3, PSMB10, PSMC4, PSMD3, PTK6, PTPN18, PTPN2, PTPN9, PYROXD2, R3HDM2, RAB29, RAB30, RAB33B, RAB6B, RAB8B, RAD18, RAET1E, RAG2, RASGRP4, RASSF1, RBM12, RBM46, RCBTB2, RCC1L, RDX, RECQL4, REEP4, REG3A, RET, RGCC, RILP, RIMS2, RIN1, RINL, RITA1, RMND5A, RNF181, RPA3, RPL18, RPL28, RPS6KA2, RRM2, RRP15, RTN2, RUFY4, RUNDC1, SAMD13, SAMD4A, SCEL, SCGB2A2, SDCBP, SDHAF2, SEC23B, SEC63, SEMA3D, SEMA6D, SENP5, SEPTIN6, SEPTIN7, SERPINA10, SERPINE2, SFT2D2, SGCA, SGCB, SGIP1, SH3GL2, SHFL, SHOC2, SIAH2, SIGLEC7, SIRPG, SIRT6, SLC17A4, SLC22A5, SLC23A1, SLC23A3, SLC25A25, SLC25A3, SLC25A40, SLC25A41, SLC27A2, SLC27A3, SLC29A4, SLC2A8, SLC37A2, SLC39A12, SLC41A3, SLC44A5, SLC46A3, SLC51B, SLC6A5, SLCO4A1, SMAD3, SMG9, SMIM19, SMOX, SMPDL3B, SNF8, SNX5, S0CS6, SOD3, SOHLH2, SOX6, SOX8, SOX9, SPAG5, SPATA4, SPATCI, SPNS1, SPOCK1, SPRED1, SPSB1, SRF, SRM, SRPK2, SRRD, SSH3, SSRP1, ST6GALNAC6, ST7, ST8SIA3, STAT4, STIMATE, STING1, STOML2, STX1B, STX5, STXBP2, SYCE1, SYT11, SYT9, TAB2, TAMM41, TBC1D21, TBCD, TBL3, TBRG4, TBX10, TBX21, TBX22, TCF7L2, TERF2IP, TES, TEX10, TEX2, TEX45, TFAP2A, TFF2, TGFB3, THOC1, THOC3, THOP1, THRB, THRSP, THUMPD3, TIPRL, TLE1, TLE4, TMED3, TMEM107, TMEM183A, TMEM64, TMEM98, TMLHE, TMOD4, TNFSF15, TNNT2, TOMM22, TPRG1L, TPST2, TRAF3IP1, TRIM40, TRIM47, TRIP13, TRMT13, TRMT1L, TRMT2A, TRPV2, TRPV5, TSGA10, TSPAN16, TSPAN2, TSPAN32, TSPAN4, TSPAN5, TUBAL3, TUBB, TUBB3, TUBB4A, TUBGCP2, TULP4, TUT7, TXNIP, U2AF1L4, UBA6, UBASH3B, UBE2B, UBE2G1, UBE2I, UBE2O, UBQLN2, UBR2, UGDH, ULK3, UMOD, UNC13D, UQCRC2, UQCRFS1, USP10, USP28, USP33, UVRAG, VAX2, VEGFA, VNN1, VNN3, VPS72, VRTN, VSIG8, VTI1B, VWC2, WBP11, WDFY2, WDR4, WDR60, WDTC1, WEE1, WFDC13, WFDC6, WNT2B, WRAP73, WSB1, XKR6, XPO7, XRCC5, XXYLT1, YAP1, YBX3, YY1AP1, ZBTB18, ZBTB39, ZDHHC20, ZDHHC5, ZIK1, ZKSCAN1, ZNF101, ZNF16, ZNF175, ZNF224, ZNF25, ZNF334, ZNF350, ZNF462, ZNF485, ZNF519, ZNF653, ZNF677, ZNF71, ZNF761, ZNF776, ZNF785, ZNHIT2, ZSCAN18 Embodiment 52a. The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment.
Embodiment 53a. The method of Embodiment 52a, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, ALOX15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, Clorf35, Clorf87, C1QB, C1QL3, C1QTNF12, C3AR1, C7orf26, CACNG3, CACNG6, CALHM1, CALHM3, CAPN2, CAPZA2, CARD8, CASD1, CASK, CATSPER2, CAVIN3, CCDC106, CCDC121, CCDC153, CCDC68, CCDC70, CCDC93, CCL28, CCNL1, CCNY, CCT7, CD300C, CD300LD, CD96, CDC37, CDC42EP2, CDCA2, CDH13, CDH26, CDHR1, CDPF1, CDRT4, CEP120, CEP43, CERKL, CERS2, CES2, CFAP161, CFAP46, CGGBP1, CHID1, CHKA, CHRND, CIART, CITED2, CITED4, CLDN18, CLEC2D, CLSTN1, CLSTN3, CNDP2, COL6A2, COLCA2, COMT, COPZ2, CORO6, CPA6, CPT1C, CREB3L2, CRLF3, CRP, CSH1, CYP2C8, CYP2D6, CYP51A1, DBT, DCDC2, DCP2, DCUN1D5, DDB1, DDI2, DDX1, DDX53, DEAF1, DELEI, DEXI, DGKG, DHRS13, DKK4, DNAJB8, DNAJC11, DNTT, D0K5, DPP4, DPYSL4, DPYSL5, DSCC1, DUSP8, DYDC2, E2F2, EBP, ECU, EDN3, EGLN3, EIF1AY, EIF2AK1, ELAVL1, ELN, ELOA, ELOA2, EMC3, EMC4, ENKUR, EN0X1, EPB41L1, EPB41L4A, EPHX4, ERICH1, ESRP1, ETFDH, FAM161B, FAM207A, FAM20C, FAM76B, FAM78A, FBXL3, FBXO31, FBXO4, FCGR2A, FCN2, FCRL5, FCRLB, FGF18, FGFR1OP2, FGL1, FHL5, FIGNL1, FLU, FNDC9, FOXA3, FPGS, FREM1, FRMD8, FSD1L, FUZ, FXR1, GABBR2, GABRQ, GATAD1, GDF10, GDF5, GDF6, GGA1, GGT5, GID8, GJA10, GKAP1, GMCL1, GNA12, GNAI3, GNB5, GNL1, GNLY, GORASP1, GORASP2, GPC5, GPN2, GPR15, GPRASP2, GRIK2, GRINA, GRP, GSPT2, GTF2F1, GTPBP2, GXYLT1, H3C10, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HIC2, HINT1, HLA-DOB, HLA- DQB2, HOXB9, H0XD3, HTR1A, HYLS1, IDS, IFNA10, IFNL3, IGFBP1, IGFBP4, IGHG1, IGHV7-81, IK, IL13RA1, IL17C, IL1R2, IL2RG, IMPA1, INKA2, INO80E, INPP5J, IQCG, IQUB, ITGB7, ITLN1, ITPA, JADE1, JAKMIP1, JAML, KCNAB2, KCND1, KCNMB1, KCTD10, KCTD13, KHDRBS3, KIAA0895, KIFC2, KLC2, KLHDC1, KLHL9, KLK1O, KPTN, KRT79, KRTAP10-7, KRTAP4-4, L3MBTL4, LAMB3, LCN9, LDHA, LHX2, LIAS, LIMA1, LIMD1, LMNA, LMO2, LOXHD1, LPCAT2, LRFN5, LRP11, LRP3, LRRC18, LRRC45, LRRFIP2, LRTM1, LYPD1, LYPD5, LYPD6B, MACROD1, MACROH2A1, MAFF, MAGEB6, MAGEH1, MALT1, MANF, MAP6D1, MAPK15, MAPKAPK5, MAPRE3, MARCKS, MARS1, MAST2, MAST3, MAST4, MC3R, MCAM, MCM8, MCM9, MCU, MDGA2, METTL25, MGAT4B, MICU2, MIDN, MINDY1, MIPEP, MLST8, MMD, MMP11, MMUT, MORN1, MOXD1, MPND, MPP2, MPV17L2, MPZL1, MRAP2, MRM3, MRPL3, MRPL4, MRPL41, MRPL48, MRPL51, MRPS30, MRS2, MSMP, MSX1, MSX2, MTMR1, MTPAP, MUC3A, MVB12A, MX1, MXRA8, MYBL1, MYO1A, MYOC, MYOM3, NABP2, NAF1, NAGLU, NAIF1, NAXD, NCCRP1, NCDN, NDOR1, NDUFA7, NDUFS8, NELLI, NEUROG3, NFE2L3, NFIA, NFS1, NHLRC3, NINJ2, NIPAL3, NKAP, NMD3, NONO, NPDC1, NPNT, NR5A1, NRSN2, NTN5, NUBP1, NUDT3, NUDT9, NUP54, NUP62, NUP85, NXNL2, NXPE3, NYX, OAS1, ODF3, ODF3L2, OGGI, OLIG1, OLIG3, OPCML, OR10H1, OR4A15, OR4K17, OR51E1, OR5D14, OR8B12, ORC3, OSGIN1, OSTN, OTUB2, OTUD5, OXGR1, P2RY12, PACC1, PACSIN1, PACSIN2, PAK4, PARG, PARM1, PAX9, PCDH8, PCDHA10, PCDHB13, PCDHB2, PCSK7, PCYOX1L, PDE4A, PDE9A, PDHB, PDIA3, PDZD7, PEMT, PENK, PEX16, PFDN5, PFKFB3, PFKFB4, PFKL, PGRMC1, PHACTR3, PHF7, PICALM, PIGH, PIGP, PKM, PKP1, PLA2G3, PLAT, PLCG2, PLD6, PLEKHG5, PLEKHO2, PLP2, PLPPR2, PLXDC2, PMFBP1, PNMA2, PNOC, PNPT1, PGDN, POGLUT3, POLR3E, POLR3F, POU3F2, PPCDC, PPP1R12C, PPP2R1A, PPP2R2D, PPP3R2, PRDM1, PRKCD, PRLHR, PRMT2, PRNP, PRPF4, PRPF40A, PRR18, PRSS22, PSMA2, PSMD5, PTDSS1, PTGES2, PTK6, PTPN18, PTPRH, PTPRJ, PTPRS, PUM3, PWP1, PXMP4, PYROXD2, QPRT, RAB23, RAB39A, RAB42, RAB6B, RAD51B, RAFI, RALGPS1, RASSF2, RBM24, RBM3, RBM34, RBM46, RCC1L, RCN3, RDH10, RHBDD1, RHOH, RHOXF1, RILPL2, RIN1, RING1, RINL, RITA1, RNF126, RNF141, RNF144B, RNF220, RNF6, RO60, ROR1, RP2, RPH3AL, RPL30, RPL34, RPL6, RRS1, RSAD2, RTL8A, RUNDC1, RUNDC3A, RUSC1, RXFP3, SAFB2, SASS6, SBK1, SCG3, SCNN1A, SDF2L1, SDHAF2, SEC14L2, SEC61G, SEC63, SEL1L2, SEMA3G, SENP5, SERBP1, SERPINB5, SF1, SFRP1, SFT2D2, SFXN3, SGK1, SGO1, SH3GL2, SH3GLB1, SHH, SHKBP1, SHOC2, SIVA1, SKIL, SLC12A1, SLC1A7, SLC22A13, SLC22A7, SLC25A20, SLC25A43, SLC27A6, SLC2A13, SLC2A8, SLC37A3, SLC45A2, SLC49A4, SLF1, SMAD6, SMARCE1, SMG5, SNAP91, SNAPC2, SNRPB, SNRPG, SNX14, SNX27, SOWAHA, SPAG5, SPATS2, SPIRE1, SPRTN, SPTLC2, SRC, SRF, SSH3, SSX3, ST3GAL6, STARD7, STIM1, STK3, STK35, STMN1, STOML3, STX10, STXBP4, SUSD3, SUSD6, SYP, TAC1, TAFA5, TBC1D19, TBCK, TBL3, TBRG4, TCAF1, TCEA2, TCF19, TCP11, TCP11L1, TDG, TDP2, TEX13A, TGM4, TGOLN2, THAP11, THBD, THOC5, TIMM29, TLR2, TM4SF4, TMED6, TMEM106B, TMEM178B, TMEM204, TMEM234, TMEM263, TMEM41A, TMEM86A, TMIGD2, TMPRSS1 IE, TMPRSS2, TNFAIP1, TNFAIP8L1, TNFRSF10C, TNFSF13B, TOR1B, TOX2, TPD52L2, TPM4, TPO, TPSD1, TPT1, TRAPPC10, TRAPPC8, TRIM40, TRIM55, TRIR, TRMT12, TRPC5, TRPV2, TSKS, TTC12, TTLL7, TUBA1C, TUBGCP2, TXNDC5, UBAC1, UBXN2A, UGT3A1, UIMC1, UNCI 19, UQCRFS1, USH1C, USP15, USP21, VAC14, VEGFA, VPS37B, VPS45, VRTN, WAPL, WDR1, WDR24, WDR54, WDR5B, WDR60, WDR61, WIPI2, WNT2, XAF1, YBX2, ZAP70, ZBTB5, ZC2HC1A, ZC3H3, ZCCHC8, ZDHHC1, ZDHHC13, ZFP2, ZFP36L2, ZGRF1, ZMPSTE24, ZNF175, ZNF19, ZNF205, ZNF274, ZNF428, ZNF436, ZNF502, ZNF558, ZNF624, ZNF668, ZNF71, ZNF710, ZNHIT2, and ZSWIM1.
Embodiment 54a. The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
Embodiment 55a. The method of Embodiment 54a, wherein the immunosuppressive resistance gene is selected from the group consisting of AARSD1, ABCC10, ABHD5, ABI1, ACAD 10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, AD ATI, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1, B9D2, BAB AMI, BAG5, BCHE, BLK, BMPR1B, BPIFC, BRF2, BSND, BTNL3, BYSL, C10orf82, C18orf25, C18orf54, Clorfll5, Clorf43, Clorf56, C1QTNF2, C1R, C2CD2, C5orfl5, C6orfl20, CAB, CA5B, CA8, CA9, CABS1, CALCOCO1, CALCR, CALHM3, CAMK2A, CAMLG, CARD8, CASP1, CASP7, CASTOR1, CBX7, CCDC110, CCDC69, CCDC82, CCDC84, CCL2, CCL21, CCR8, CCSER1, CD151, CD300LF, CD48, CD83, CD86, CDH7, CDK1, CDPF1, CDR2, CELF1, CEP63, CERS1, CES3, CFAP410, CGGBP1, CHAF1B, CHMP2A, CHMP7, CHRM1, CHST9, CISH, CLC, CLDN6, CLEC5A, CLIC5, CLP1, CLTRN, CLUAP1, CMTM7, CNTF, C0G3, COL25A1, COL8A2, C0MMD4, C0PZ1, COPZ2, COQ4, COX6B2, CPLX2, CRACR2B, CROT, CRTAC1, CRY2, CSF1, CSNK1G2, CST9, CSTF3, CTDP1, CTNNA2, CTSF, CXCR6, CXXC1, CYP27A1, CYP2D6, CYTL1, DAXX, DBN1, DDHD2, DDX24, DES, DEXI, DGLUCY, DHX36, DNAI2, DNAJA2, DNAJB5, DNAJB6, DNAJC11, DNAJC27, DNAJC6, D0K5, D0K6, DRGX, DUS1L, DUSP5, DZIP1L, ECI2, EFCAB7, EGFL6, EGLN3, EIF3K, EIF4EBP1, ELSPBP1, EMC3, EPDR1, EPN1, EPO, ERFE, ERVK3-1, ESMI, F2RL2, FAAP100, FADS1, FAM172A, FAM53C, FAM71C, FAM81A, FBLN5, FBXL16, FBXO7, FBXW11, FCGR3A, FCRL5, FDFT1, FETUB, FEZF2, FGF10, FGF19, FGL1, FKBP5, FKBP9, FMOD, FNDC9, FOSB, FRMD3, FRMD5, FRZB, FUT3, GAB3, GABRA4, GABRG2, GALNT2, GALNTL6, GAS7, GBA2, GBP6, GEMIN8, GET1, GFM2, GFRA3, GK2, GLIPR1, GLRA2, GLRB, GLYCTK, GNA14, GNB3, GNL3, GOLM2, GPATCH2L, GPATCH3, GPM6B, GPR176, GPR45, GRAMD1B, GRB7, GRK2, GRK7, GSTM3, GXYLT1, GZMM, HAPLN3, HAUS2, HBG2, HCRTR1, HDAC8, HDGF, HEMK1, HERPUD1, HESX1, HEXA, HMHB1, HOXB5, HOXB6, HOXD3, HOXD4, HPGDS, HSD17B6, HSD17B8, HSPA2, HTN1, HTR5A, IFITM3, IFNA10, IGF1, IGFALS, IGHM, IL11RA, IL17A, IL17RE, IL2RB, IL4, INSL6, ISL2, ISM2, IST1, ITPRID2, JAML, JUN, JUNB, KBTBD7, KCNA6, KCNN3, KEAP1, KIF3A, KIFC2, KIR2DL1, KLHDC2, KLHDC7B, KLHL9, KRT19, KRT6A, LARS2, LCK, LCN1, LDAH, LDLRAD4, LETMD1, LHX4, LHX9, LIN28A, LIPG, LNX1, LPAR5, LPL, LRFN3, LRRC15, LRRC2, LRRC34, LRRC42, LTBR, LYZ, MAF1, MAGOH, MAN1A1, MAN1B1, MAP2K6, MAP3K7, MAP4K5, MAPKAPK2, MAPKAPK5, MARCHF1, MARCHF2, MBNL1, MBP, MC3R, MCAM, MED26, MEIS3, METTL27, METTL2B, MIA2, MIF, MIPOL1, MLKL, MMD, MMP10, MOB4, MPHOSPH8, MPND, MPZL1, MR1, MRAS, MRM3, MRPL21, MRPS24, MRPS28, MS4A3, MS4A5, MSRA, MTA2, MTA3, MTHFD2, MTMR3, MTPAP, MTRF1L, MUS81, MYCL, MYCN, MYL10, MYL9, MYOC, MY0M3, MYORG, NAA80, NAPSA, NARF, NAT1, NCK1, NDE1, NDOR1, NDUFA13, NDUFA4, NEK5, NELFE, NFIB, NFKBIB, NINJ2, NIPAL1, NKAIN2, NKAP, NMB, NOC4L, NPIPB15, NRARP, NRSN2, NTF3, NXNL2, OBP2A, OLR1, OR10AG1, OR10K2, OR14C36, OR1F1, OR2M3, OR2T8, OR5C1, OR7A5, OR9Q1, ORAI3, ORC2, ORM1, ORM2, OSGIN1, OSR2, OTUD5, P3H4, P4HA3, P4HTM, PAK2, PAK4, PBX2, PCDHA2, PCDHB12, PCDHGA2, PDYN, PFDN4, PFKP, PFN4, PGK1, PGLYRP1, PHF23, PHF7, PHKG1, PH0SPH01, PI15, PI3, PI4KB, PITHD1, PKIA, PKNOX2, PLA2G3, PLA2G7, PLAUR, PLEKHA8, PLEKHO2, PLET1, PLOD2, PNPT1, POPDC3, PPM1D, PPME1, PPP1R2, PPP1R32, PPP2R2B, PPP2R3C, PPP2R5C, PPP6R2, PRKCB, PRKRIP1, PRKY, PRMT8, PRSS3, PRUNE2, PSCA, PSG1, PTGER3, PTK6, PTOV1, PTPRO, PTTG1IP, PUDP, PWWP3B, PYCARD, PYGB, QPCT, QPRT, RABI IB, RAB25, RAB28, RAB34, RAB40B, RABEP2, RADU, RAET1E, RAMP1, RARS1, RAX2, RBBP5, RCHY1, RCN3, REEP2, RFC4, RFPL2, RFX3, RIBCI, RINL, RIPK4, RLBP1, RNASE9, RNASET2, RNF111, RNF112, RNF144B, RNF24, RNF38, RNF7, ROPN1L, RP9, RPL6, RPS2, RPS3A, RPS6, RRAGA, RRAGD, RRP1, RRP9, RSAD1, RUBCN, RUNX1T1, RUVBL1, SAMHD1, SARS1, SCNN1A, SCNN1B, SCNN1G, SEL1L2, SELENBP1, SEPHS1, SEPTIN10, SEPTIN12, SERINC2, SERPINA3, SERPIND1, SERPINE1, SERPINE3, SETD3, SFRP4, SGK2, SH3KBP1, SHARPIN, SHISA3, SHOC2, SHOX, SIGLEC7, SIRPB2, SIRPG, SIRT3, SKIL, SLC13A1, SLC14A1, SLC20A2, SLC22A13, SLC22A31, SLC22A8, SLC25A1, SLC25A46, SLC25A47, SLC25A48, SLC2A8, SLC37A3, SLC39A7, SLC5A12, SLC6A19, SLC6A7, SLC7A9, SLCO1A2, SLFNL1, SMG9, SMPX, SNORC, SNRNP25, SNRPB, SNRPN, SNX16, SOAT1, SOCS5, SPATA22, SPATS2, SPC25, SPG21, SPINT1, SPINT2, SPNS2, SPP2, SQOR, SRC, SRFBP1, SRP54, SRP9, SRSF9, SSBP2, SSPN, STAP1, STARD7, STIM1, STK11, STX8, SULT4A1, SUMF2, SURF6, SYMPK, SYNCRIP, SZT2, TAS2R40, TAS2R60, TBCC, TBRG4, TBX20, TBX3, TON, TCEA1, TCEA2, TCF19, TCF7L2, TCN2, TCTN1, TDP2, TENT5C, TEX35, TFCP2L1, TFDP2, TGFB3, THAP12, TIAM2, TICAM2, TIPIN, TKT, TLE4, TM2D2, TM9SF3, TMEM106B, TMEM143, TMEM160, TMEM211, TMEM237, TMEM270, TMEM30A, TMEM39B, TMEM45A, TMEM68, TMPRSS3, TNFSF12, TOMM70, TOR1AIP1, TOX, TPP1, TPRKB, TPST2, TRAPPC12, TRDMT1, TRIM10, TRIM47, TRIM63, TRIP10, TRMO, TRMT44, TRPV4, TSN, TSPAN31, TSPEAR, TSSK3, TTBK2, TTC32, TUBA3C, TUBA3D, TUT7, TYW3, UBA1, UBASH3B, UBE2S, UBXN2A, UCHL3, UCHL5, UQCR10, USP1, USP19, VAT1L, VMA21, VPS29, VPS36, VSTM2A, VWA1, WAS, WDFY1, WDR4, WDR59, WDR63, WDR78, WNT11, WNT3A, WNT9A, YAF2, YJU2, ZBTB48, ZC3HAV1L, ZCCHC2, ZCCHC7, ZFP36L2, ZIC3, ZNF165, ZNF830, ZP2, ZSCAN21, and ZSCAN9. Embodiment 56a. The method of Embodiment 48a, wherein the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment.
Embodiment 57a. The method of Embodiment 56a, wherein the immunosuppressive resistance gene is selected from the group consisting of AB AT, AB HD 12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, ALOXE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GALNT2, B3GALT4, B3GNT2, BAB AMI, BAP1, BCAP31, BCCIP, BCL2L2, BCR, BDNF, BECN1, BEND2, BEND7, BLOC1S4, BMP5, BMPR1B, BRD3, BRF2, BRINP3, BTBD17, BTNL3, C10orf62, C12orf42, C14orf28, Clorf210, C2orf78, C6, C7orf31, CAB, CACNB3, CALR3, CARD14, CARD19, CASC1, CASP10, CASQ2, CBFB, CBX3, CBX4, CCDC141, CCDC148, CCDC68, CCDC8, CCDC82, CCIN, CCNG2, CCNY, CCR1, CCR10, CCR3, CD19, CD244, CD47, CD5L, CD83, CDC42EP1, CDC42EP4, CDCA2, CDCP2, CDH13, CDYL2, CEP120, CEP63, CERCAM, CERKL, CFAP100, CFAP20, CFAP410, CFAP47, CFAP91, CFHR1, CHGA, CHUK, CIRBP, CLCNKB, CLDND1, CLU, CMTR1, CNPPD1, COA3, COG1, COL22A1, COL25A1, COL6A2, COL8A2, COPB1, COPS3, COQ3, COQ8B, COX18, CPOX, CPQ, CPT1A, CPXM1, CRACR2A, CREB3L1, CRNN, CRTAP, CRYGD, CS, CSF1, CSF1R, CSF2RB, CSF3R, CSGALNACT1, CSTF2, CTCF, CTSV, CUEDC1, CUL2, CXADR, CYB5R2, CYP20A1, CYP26A1, CYP2C19, CYP8B1, DAAM2, DAB1, DACT2, DARS1, DBN1, DCAF4L2, DCAF8, DCT, DDO, DDR1, DDX43, DDX54, DEAF1, DECR1, DENR, DESI2, DHX40, DIP2A, DKK4, DNAAF3, DNAI2, DNAJC7, DNAL1, DPP4, DPT, DPYSL4, DRG1, DSCAM, DTL, DTX2, DUS1L, DUSP12, E2F7, ECHI, EFEMP2, EFHC1, EFS, EIF2B3, EIF4A2, EIF5, ELAVL4, ELL, ELMOD3, ELN, ELOA, ELOA2, ELP3, EMC7, ENCI, ENDOV, ENPP3, ENTPD5, EOLA2, EPB41L1, EPHB6, EPN1, ERCC6, ESRP1, ESRRA, ETFDH, EXO1, EXOC3, EXOSCIO, EXOSC8, EYS, F3, FABP6, FAM117A, FAM117B, FAM118B, FAM161B, FAM200A, FAM20A, FAM47A, FARSA, FBLN1, FBLN5, FBP1, FBXO24, FBXO40, FCHO1, FCRL5, FCRLB, FECH, FEM1B, FGF3, FGG, FKBP6, FLVCR1, FMNL1, FM05, FNIP1, FOS, FOSB, FOXD4, FOXJ1, F0XM1, FOXN3, FOXRED2, FREM1, FRMD5, FRS2, FSTL1, FSTL4, FSTL5, FTMT, FXR2, G6PD, GABPB1, GABRB1, GALNT7, GANAB, GAPDHS, GATA2, GBP4, GCAT, GCGR, GCNT7, GCSAML, GDF2, GDF6, GET4, GGA1, GHDC, GINS4, GK, GKAP1, GLB1, GLRA2, GLRX5, GLYR1, GMCL2, GNA14, GNAT2, GNB3, GNL1, GNL3, GPAT4, GPC3, GPC4, GPC5, GPR84, GRHL3, GRHPR, GRIA4, GRID1, GRIK3, GRK4, GRM3, GRM8, GRN, GSDME, GSK3A, GSN, GTF2A1, GTF2B, GTF2E1, GUCY1B1, GYSI, HABP2, HADHA, HADHB, HDAC10, HEPHL1, HERPUD2, HES1, HEXD, HHIPL2, HINT2, HLA-C, HOMER3, HOOK3, HOXB6, HOXD3, HPS3, HPS5, HSD11B2, HSD17B13, HSD17B7, HSD3B1, HSPB9, HTR2B, IFNA6, IFNL1, IFT27, IGHA1, IGSF1O, IGSF21, IL1ORB, IL12RB2, IL13RA1, IL21, IL26, IL7R, ILVBL, IMPG1, INA, INHA, INSL4, INTU, IQUB, IRAG1, IRAKI, IREB2, IRF3, IRF5, IRX3, ITFG1, ITGB2, ITGB7, ITIH5, ITM2B, JAKMIP1, KANSL3, KAT5, KCNG3, KCNJ14, KCNK12, KCNK2, KCNK9, KCNMB3, KCTD12, KCTD4, KIAA2013, KIF2C, KIF3A, KIF3B, KIFC2, KITLG, KLC3, KLHDC2, KLHL13, KLHL21, KLHL8, KLK1, KRT6A, KXD1, L3MBTL4, LAD1, LAMP1, LAP3, LARS2, LDLRAD3, LGMN, LHX2, LHX4, LIMA1, LIMD1, LIPH, LIPT1, LKAAEAR1, LNPEP, LOXHD1, LOXL3, LTA4H, LTBR, LUC7L2, LYL1, LZTS2, MAG, MAN1A1, MANEA, MANF, MAP3K7, MAP4K5, MAP6D1, MAPK15, MAPK8, MAPKAPK5, MAPKBP1, MARS2, MATN2, MBLAC1, MCAM, MCOLN2, MCOLN3, MDH1B, MDM4, MEAK7, MECP2, MED1, MEOX1, METAP1, MFAP3L, MFNG, MITD1, MLEC, MLH1, MLLT3, MMP10, MMP16, MMS19, MNAT1, MON1B, MPP2, MPP7, MRM3, MRPL19, MRPL3, MRPL47, MSLN, MSRA, MTARC2, MTHFR, MTMR4, MTMR6, MUTYH, MVP, MYBL1, MYCN, MYO1A, MYO5C, MYOC, MYOM3, MYORG, MYT1, NAGLU, NAMPT, NCKIPSD, NDC80, NDRG4, NDUFAF7, NDUFB6, NDUFS8, NDUFV2, NECTIN4, NELFA, NFIB, NIF3L1, NIPAL1, NIPBL, NMD3, NOC2L, NPLOC4, NPY, NPY2R, NQO1, NR2E1, NR6A1, NT5DC2, NTM, NTN5, NTNG1, NUDT8, NUP210, NXPE3, OAS1, OAS2, ODF2, OGA, OGFRL1, OLFM2, OLFML1, OLFML2B, OLIG1, OLR1, OMP, OR4D1, OR4S2, OR52N5, OR52W1, OR5B3, OR9Q1, ORMDL2, OSBPLIO, OSGEPL1, OTX1, OXSR1, P2RX6, P2RY1, PACS2, PAEP, PAFAH1B2, PAK4, PARL, PARP9, PARS2, PC, PCDHA1, PCDHA6, PCDHGA2, PCDHGA5, PCSK2, PDCD6, PDE4A, PDIA3, PDK2, PDZD9, PENK, PFKL, PGAM5, PGK1, PGM2, PHF11, PHYH, PICALM, PIK3R1, PIP5K1B, PLAGL1, PLCD4, PLD1, PLEK, PLEKHG5, PLEKHS1, PLIN1, PLK1, PLPPR2, PM20D1, PNMA8A, POLE, POLI, POLR2K, POLR3F, POU3F2, PPA2, PPIG, PPM1D, PPP1R12C, PPP1R16B, PPP1R32, PPP2R3C, PPP2R5C, PPP2R5D, PRAC1, PRAM1, PRAME, PRDM1, PRDX3, PRKAA2, PRKAG2, PRKAR1B, PRKCE, PRMT1, PROC, PRRT2, PRSS45P, PRSS48, PSAPL1, PSD3, PSEN1, PSMC4, PSMD14, PTBP3, PTCD2, PTDSS1, PTGER2, PTPN12, PTPRN, PUF60, PUM1, PYDC1, PYGB, RAB1A, RAB33B, RAB42, RABL6, RAD18, RAD51, RAET1G, RALBP1, RASSF4, RBBP7, RBM12, RBM38, RBM4, RBM46, RBM4B, RBX1, RCBTB2, RCC1L, RDH10, RDH12, REC8, REN, RET, RFC2, RFC5, RFX4, RGS16, RHAG, RHEX, RHOBTB2, RICTOR, RIMBP2, RIMS3, RIOK3, RIPK1, RIPK2, RIT1, RMDN3, RNASEH2B, RNF114, RNF148, RNF213, RNF6, RNPEPL1, RO60, RORA, RPL30, RPS14, RPS4X, RPS6KA2, RPS6KB1, RSRC1, RTF1, RTN1, RUBCN, RUNDC1, RUNX3, S1OOPBP, SAMHD1, SCAMP2, SCIN, SCNN1D, SCRN2, SDSL, SEC63, SEL1L3, SELL, SENP3, SEPTIN10, SEPTIN8, SERINC3, SERPINF1, SGK3, SGMS1, SH3GLB1, SHOC2, SIAH2, SIGLEC10, SIGLEC12, SIGLEC7, SIRPG, SIRT5, SKAP1, SKIL, SLC15A3, SLC16A1, SLC16A7, SLC18A2, SLC1A7, SLC20A2, SLC22A2, SLC22A23, SLC22A24, SLC23A2, SLC25A19, SLC25A25, SLC25A47, SLC26A2, SLC2A4, SLC2A8, SLC36A3, SLC37A2, SLC37A3, SLC39A14, SLC43A1, SLC5A11, SLC5A12, SLC5A7, SLC6A7, SLC7A1, SLCO1A2, SLITRK3, SMARCD3, SMOX, SNCAIP, SNTA1, SOCS6, SOX9, SPATA2, SPCS3, SPN, SPNS2, SPOCK3, SPOUT1, SPRR4, SRC, SRD5A3, SRFBP1, SRMS, SSMEM1, SSX2IP, ST7L, STEAP1, STIM1, STK11, STK17A, STK17B, STK3, STK39, STXBP1, STXBP2, STXBP3, STXBP5, SUGCT, SUN5, SUSD2, SYNCRIP, TAB2, TBC1D10A, TBC1D22B, TBC1D9B, TBCD, TBL1X, TBX6, TON, TCF25, TCTN2, TDGF1, TEAD2, TESK1, TESK2, TEX48, TFAP2A, TFAP4, TGFB1, TGFBI, TGOLN2, THAP3, THAP7, THEM4, THOC1, THOC7, THOP1, THSD4, TIAM2, TICAM2, TLE6, TLK1, TM9SF4, TMED2, TMEM259, TMEM62, TMIE, TMLHE, TMOD1, TMTC3, TNF, TNS1, TOMM70, TRAF3IP1, TRAM1, TRAP1, TRAPPC3, TRAPPC4, TRAPPC8, TRAPPC9, TRIB3, TRIM32, TRMT13, TRPM3, TRPV4, TSGA10, TSPEAR, TSPYL6, TTC13, TTC16, TTC26, TTC38, TTC8, TTF2, TTLL2, TTLL7, TUBA3E, TUBA8, TUBB3, TUBB4B, TUFM, TYMS, UBE2O, UBE2Z, UBE3A, UBE3C, UBOX5, UGDH, UGP2, UGT3A1, UGT8, UMOD, USP1, USP49, UXS1, VANGL2, VASN, VEGFA, VIL1, VPS45, VWA1, WAPL, WDR37, WDR63, WDR90, WDR91, WNT4, WWOX, XRCC5, YBX2, YME1L1, ZBTB14, ZBTB18, ZBTB46, ZC3H10, ZC3H11A, ZCCHC8, ZDHHC11, ZDHHC15, ZDHHC6, ZFYVE21, ZGRF1, ZMAT3, ZNF165, ZNF18, ZNF19, ZNF20, ZNF224, ZNF25, ZNF277, ZNF334, ZNF350, ZNF354C, ZNF396, ZNF398, ZNF436, ZNF461, ZNF467, ZNF496, ZNF560, ZNF565, ZNF566, ZNF597, ZNF610, ZNF623, ZNF668, ZNF74, ZP1, ZPLD1, ZSCAN25, and ZSWIM2.
Embodiment 58a. A method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of:
(i) collecting peripheral blood mononuclear cells (PBMCs) from an individual,
(ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i),
(iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and
(iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
Embodiment 59a. The method of Embodiment 58a, wherein the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR).
Embodiment 60a. The method of Embodiment 58a or Embodiment 59a, wherein the exogenous nucleic acid further comprises a T cell receptor (TCR).
Embodiment 61a. The method of any one of Embodiments 58a-60a, wherein the peripheral blood mononuclear cells are obtained from leukapheresis.
Embodiment 62a. The method of any one of Embodiments 58a-61a, wherein the CD8+ and CD4+ T cells are isolated sequentially.
Embodiment 63a. The method of any one of Embodiments 58a-62a, wherein the naive CD4+ T cells are differentiated into activated CD4+ T cells.
Embodiment 64a. The method of any one of Embodiments 58a-63a, wherein the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
Embodiment 65a. The method of any one of Embodiments 58a-64a, wherein the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
Embodiment 66a. The method of any one of Embodiments 58a-65a, wherein the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
Embodiment 67a. The method of any one of Embodiments 58a-66a, wherein the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
Embodiment 68a. The method of any one of Embodiments 58a-67a, wherein the transduced lymphocytes are enriched by positive selection. Embodiment 69a. The method of Embodiment 68a, wherein the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
Embodiment 70a. A method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising:
(i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual,
(ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes,
(iii) transiently stimulating the transduced lymphocytes,
(iv) exposing the transduced lymphocytes to an immunosuppressive environment,
(iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and
(v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene.
Embodiment 71a. The method of Embodiment 70a, wherein the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.

Claims

1. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of MCAM, FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
2. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABI1, ACSL4, ACSM3, ADARB1, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, A0C1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, C0R06, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, D0K5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EX01, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FM05, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, L0XHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
3. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene selected from the group consisting of CD47, CD86, COPZ2, FOSB, GNL3, GSDME, IL12RB2, IL26, LIMA1, LTBR, MCAM, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZBTB46; wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof; and wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein.
4. The modified lymphocyte of any one of claims 1-3, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding the immunosuppressive resistance gene.
5. The modified lymphocyte of any one of claims 1-4, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding the immunosuppressive resistance gene.
6. A modified lymphocyte that is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, wherein the modified lymphocyte further comprises a nucleic acid encoding a therapeutic protein, and wherein the modified lymphocyte has increased proliferation and/or increased effector function in in vivo or in vitro tumor killing assays relative to an unmodified lymphocyte.
7. The modified lymphocyte of claim 6, wherein the modified lymphocyte comprises an exogenous nucleic acid encoding LTBR.
8. The modified lymphocyte of claim 6 or claim 7, wherein the modified lymphocyte comprises an expression cassette comprising a promoter and the nucleic acid encoding LTBR.
9. The modified lymphocyte of any one of claims 3 and 6-8, wherein the increased level of the immunosuppressive resistance gene results in increased tumor cell killing of the modified lymphocyte in a tumor killing assay compared to a lymphocyte that does not express the immunosuppressive resistance gene.
10. The modified lymphocyte of any one of claims 1-9, wherein the nucleic acid further encodes at least two immunosuppressive resistance genes selected from the group consisting of FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, and VEGFA.
11. The modified lymphocyte of any one of claim 1-10, wherein the modified lymphocyte expresses the therapeutic protein.
12. The modified lymphocyte of any one of claims 1-11, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
13. The modified lymphocyte of claim 12, wherein the CAR or the TCR binds to a tumor antigen.
14. The modified lymphocyte of any one of claims 6-8, wherein the increased level of LTBR results in increased killing of cells expressing a low level of antigen in a tumor killing assay compared to a lymphocyte that does not express the LTBR, wherein the therapeutic protein is a CAR or a TCR, wherein the antigen is bound by the CAR or the TCR.
15. The modified lymphocyte of claim 14, wherein the tumor killing assay is an in vitro tumor killing assay.
16. The modified lymphocyte of any one of claims 12-15, wherein the CAR or the TCR binds to HER2 or Claudin-6 (CLDN6).
17. The modified lymphocyte of any one of claims 12-16, wherein the modified lymphocyte further comprises an expression cassette comprising a nucleic acid encoding the CAR or the TCR.
18. The modified lymphocyte of claim 17, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or the TCR are located in the same expression cassette.
19. The modified lymphocyte of claim 17, wherein the nucleic acid encoding the immunosuppressive resistance gene and the nucleic acid encoding the CAR or TCR are located in separate expression cassettes.
20. The modified lymphocyte of any one of claims 1-19, wherein exhaustion of the modified lymphocyte is reduced compared to a lymphocyte that does not express the immunosuppressive resistance gene.
21. The modified lymphocyte of any one of claims 1-20, wherein the modified lymphocyte maintains the ability to kill tumor cells expressing a tumor antigen following at least two exposures to the tumor cells.
22. The modified lymphocyte of any one of claims 1-21, wherein the modified lymphocyte is a T cell, a NK cell, or a NK T cell.
23. The modified lymphocyte of any one of claims 1-22, wherein the modified lymphocyte is derived from induced pluripotent stem cells (iPSCs).
24. The modified lymphocyte of any one of claims 1-23, wherein the modified lymphocyte is a tumor infiltrating lymphocyte (TIL).
25. The modified lymphocyte of any one of claims 1-24, wherein the modified lymphocyte is a CD4+ T cell or a CD8+ T cell.
26. The modified lymphocyte of any one of claims 1-24, wherein the modified lymphocyte is a TCRaP+ CD4- CD8- T cell.
27. The modified lymphocyte of any one of claims 1-24, wherein the modified lymphocyte is a TCRy8+ lymphocyte.
28. The modified lymphocyte of claim 27, wherein the modified lymphocyte is a TCRy8+ lymphocyte and is engineered to express an increased level of an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene is LTBR.
29. The modified lymphocyte of claim 28, wherein the increased level of the immunosuppressive resistance gene LTBR results in increased killing of antigen-low cancer cells in tumor killing assays compared to an unmodified lymphocyte.
30. The modified lymphocyte of claim 29, wherein the tumor killing assay is an in vitro tumor killing assay.
31. The modified lymphocyte of any one of claims 1-24, wherein the modified lymphocyte is a naive T cell, a stem cell-like memory (TSCM) T cell, a central memory (TCM) T cell, an effector memory (TEM) T cell, or an effector memory RA+ (TEMRA) T cell.
32. The modified lymphocyte of any one of claims 1-24, wherein the modified lymphocyte is a regulatory T cell.
33. The modified lymphocyte of any one of claims 1-32, wherein the modified lymphocyte is a human lymphocyte.
34. The modified lymphocyte of any one of claims 1-33, wherein the modified lymphocyte is an autologous lymphocyte.
35. The modified lymphocyte of any one of claims 5-34, wherein the modified lymphocyte comprises a vector comprising the expression cassette.
36. The modified lymphocyte of any one of claims 5-35, wherein the expression cassette comprises a promoter that is operably linked to the immunosuppressive resistance gene.
37. The modified lymphocyte of claim 36, wherein the promoter is a ubiquitous promoter.
38. The modified lymphocyte of claim 37, wherein the ubiquitous promoter is selected from the group consisting of cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor la (EFla), ubiquitin C (UbC), 5’LTR, and CMV.
39. The modified lymphocyte of claim 36, wherein the promoter is an inducible promoter.
40. The modified lymphocyte of any one of claims 36-39, wherein the promoter drives constitutive expression of the immunosuppressive resistance gene in the modified lymphocyte.
41. The modified lymphocyte of any one of claims 36-40, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the genome of the modified lymphocyte.
42. The modified lymphocyte of any one of claims 36-41, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into the native genomic locus of the immunosuppressive resistance gene.
43. The modified lymphocyte of any one of claims 36-41, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is integrated into a safe harbor locus.
44. The modified lymphocyte of any one of claims 36-41, wherein the expression cassette comprising the nucleic acid encoding the immunosuppressive resistance gene is randomly integrated into the genome of the modified lymphocyte.
45. A vector comprising nucleic acid encoding an immunosuppressive resistance gene, wherein the immunosuppressive resistance gene encodes a full-length protein or a functional fragment thereof, and wherein the immunosuppressive resistance gene is selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, HOXD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, VEGFA, ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS IL, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EX01, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, H0XB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLCO1A2, SMG9, SMOX, SNRPB, SOCS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
46. The vector of claim 45, wherein the vector further comprises an expression cassette comprising a promoter that is operably linked to the nucleic acid encoding the immunosuppressive resistance gene.
47. The vector of claim 45 or claim 46, further comprising nucleic acid encoding a therapeutic protein.
48. The vector of claim 47, wherein the therapeutic protein comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
49. The vector of claim 48, wherein the nucleic acid encoding the CAR or the TCR is included in the same expression cassette as the nucleic acid encoding the immunosuppressive resistance gene.
50. The vector of claim 48, wherein the nucleic acid encoding the immunosuppressive resistance gene is included in a first expression cassette and the nucleic acid encoding the CAR or the TCR is included in a second expression cassette.
51. The vector of any one of claims 45-50, wherein the vector is a viral vector.
52. The vector of any one of claims 45-51, wherein the vector is a lentivirus, an adenovirus, a retrovirus, or a baculovirus.
53. The vector of claim 52, the vector is an episomal or non-integrating vector.
54. The vector of claim 53, wherein the episomal vector is a Simian virus 40 (SV40), Adenovirus, or Adeno-associated vector.
55. The vector of any one of claims 45-50, wherein the vector is a non-viral vector.
56. The vector of claim 55, wherein the non-viral vector is a plasmid.
57. The vector of any one of claims 45-56, wherein the vector further comprises nucleic acid encoding a drug-resistance gene, an intracellular enzyme, a fluorescent protein, and/or a surface expressed safety switch gene.
58. A modified lymphocyte comprising one or more of the vectors according to any one of claims 45-57.
59. A composition comprising the modified lymphocyte according to any one of claims 1-44.
60. The composition of claim 59, wherein the composition comprises a mixture of CD4+ and CD8+ T cells engineered to express an increased level of one or more immunosuppressive resistance genes.
61. A method of increasing lymphocyte proliferation in an immunosuppressive cellular environment, comprising introducing into lymphocytes the vector of any one of claims 44-56 or increasing expression of an immunosuppressive resistance gene selected from the group consisting of: FOSB, COPZ2, DNAI2, DPYSL4, ESRP1, FAM161B, FCRL5, FCRLB, FRMD5, GDF6, GGA1, GKAP1, GPC5, H0XD3, KIFC2, LARS2, LHX4, LTBR, MAP6D1, MAPKAPK5, MCAM, MRM3, MRPL3, MYBL1, MY01A, MYOC, MY0M3, MYORG, NXPE3, PAK4, PDE4A, PFKL, PLEKHO2, POU3F2, PPP1R32, PTK6, RBM46, RCC1L, RINL, RUNDC1, SEC63, SHOC2, SIGLEC7, SIRPG, SKIL, SLC2A8, SLC37A3, SRC, STIM1, TBRG4, VEGFA, ABCE1, ABI1, ACSL4, ACSM3, AD ARBI, ADGRG7, ALG3, ALK, ALKBH5, AMY2B, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, AQP1, ARFGAP1, ARMC2, ASAP3, ASB3, ATP5F1B, ATP6V0C, AUTS2, BAB AMI, BAP1, BCAT1, BCKDHA, BCL2L2, BMPR1B, BRF2, BTNL3, C18orf54, C1QTNF12, CAB, CALCOCO1, CALHM1, CALHM3, CARD8, CCDC121, CCDC68, CCDC82, CCDC93, CCIN, CCNY, CCT7, CD19, CD83, CD86, CD96, CDCA2, CDH13, CDPF1, CEP120, CEP63, CERKL, CFAP410, CFAP46, CFAP47, CGGBP1, CLSTN1, CNDP2, COL22A1, COL25A1, COL6A2, COL8A2, CORO6, CREB3L2, CRYGD, CSF1, CTNNA2, CYP20A1, CYP2D6, DAXX, DBN1, DCDC2, DDB1, DDO, DEAF1, DEXI, DKK4, DNAJC11, DOK5, DPP4, DUS1L, E2F7, ECU, EFEMP2, EGLN3, ELN, ELOA, ELOA2, EMC3, ENPP3, EPB41L1, EPHB6, EPN1, ETFDH, EXO1, FAM200A, FBLN5, FBXL16, FGF3, FGL1, FKBP5, FLVCR1, FMO5, FNDC9, FOXJ1, FREM1, GAB3, GABRQ, GALNT7, GAPDHS, GBP4, GET1, GET4, GLRA2, GLRB, GNA14, GNB3, GNL1, GNL3, GORASP1, GORASP2, GPRASP2, GRID1, GRINA, GSDME, GTPBP2, GXYLT1, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HLA-C, HOXB6, HPS3, IDS, IFNA10, IGFBP1, IK, IL12RB2, IL13RA1, IL17RE, IQUB, IREB2, ITGB7, JAKMIP1, JAML, KCNG3, KCNJ14, KCNK12, KCNK9, KCTD4, KHDRBS3, KIF3A, KLHDC2, KLHL8, KLHL9, KRT6A, KRT79, L3MBTL4, LCK, LHX2, LIMA1, LIMD1, LNX1, LOXHD1, LRP3, LRRC18, LRRC45, MAG, MAN1A1, MANF, MAP3K7, MAP4K5, MAPK15, MC3R, MCOLN3, MED1, MGAT4B, MICU2, MLST8, MMD, MMP10, MPND, MPP2, MPZL1, MSRA, MTHFD2, MTPAP, MYCL, MYCN, NAGLU, NCDN, NDOR1, NDUFS8, NFIB, NIF3L1, NINJ2, NIPAL1, NKAP, NMD3, NPLOC4, NPNT, NRSN2, NTN5, NXNL2, OAS1, OLFM2, OLIG1, OLR1, OR1F1, OR9Q1, 0RM2, OSGIN1, OTUD5, OTX1, P4HTM, PAX9, PCDHGA2, PDE9A, PDHB, PDIA3, PDZD9, PENK, PFKP, PGK1, PHF7, PI3, PICALM, PLA2G3, PLA2G7, PLAGL1, PLAT, PLEKHG5, PLOD2, PLPPR2, PNPT1, POLR3F, PPA2, PPM1D, PPP1R12C, PPP1R16B, PPP2R2B, PPP2R3C, PPP2R5C, PRAME, PRDM1, PRDX3, PRKCE, PRMT8, PRSS3, PSMC4, PTDSS1, PTPN18, PYGB, PYROXD2, QPRT, RAB33B, RAB42, RAB6B, RAD18, RAET1E, RBM12, RCBTB2, RCN3, RDH10, RET, RIN1, RITA1, RNF144B, RNF6, RO60, RPL30, RPL6, RPS6KA2, RUBCN, SAMHD1, SCNN1A, SDHAF2, SEL1L2, SENP5, SEPTIN10, SFT2D2, SH3GL2, SH3GLB1, SIAH2, SLC1A7, SLC20A2, SLC22A13, SLC25A25, SLC25A47, SLC37A2, SLC5A12, SLC6A7, SLC01A2, SMG9, SMOX, SNRPB, S0CS6, SOX9, SPAG5, SPATS2, SPNS2, SRF, SRFBP1, SSH3, STARD7, STK11, STK3, STXBP2, SYNCRIP, TAB2, TBCD, TBL3, TC2N, TCEA2, TCF19, TCF7L2, TDP2, TFAP2A, TGFB3, TGOLN2, THOC1, THOP1, TIAM2, TICAM2, TLE4, TMEM106B, TMLHE, TOMM70, TPST2, TRAF3IP1, TRAPPC8, TRIM40, TRIM47, TRMT13, TRPV2, TRPV4, TSGA10, TSPEAR, TTLL7, TUBB3, TUBGCP2, TUT7, UBASH3B, UBE2O, UBXN2A, UGDH, UGT3A1, UMOD, UQCRFS1, USP1, VPS45, VRTN, VWA1, WAPL, WDR4, WDR60, WDR63, XRCC5, YBX2, ZBTB18, ZCCHC8, ZFP36L2, ZGRF1, ZNF165, ZNF175, ZNF19, ZNF224, ZNF25, ZNF334, ZNF350, ZNF436, ZNF668, ZNF71, and ZNHIT2.
62. The method of claim 61, wherein the immunosuppressive cellular environment comprises a tumor microenvironment.
63. The method of claim 61, wherein the immunosuppressive cellular environment comprises an adenosine driven immunosuppressive cellular environment.
64. The method of claim 63, wherein the immunosuppressive resistance gene is selected from the group consisting of IL12RB2, COPZ2, ABCB7, ABCE1, ABCF1, ABCG2, ABR, ACRBP, ACSL4, ACSM3, ACTG2, ACTR10, ACTRT3, AD ARBI, ADGRG5, ADGRG7, ADIRF, ADSS2, AGK, AGPAT5, AGTR1, AIMP1, AK6, AKAP10, AKAP13, AKAP14, AKIP1, ALDH6A1, ALDOB, ALG5, ALKBH5, AMY2B, ANGPT1, ANKEF1, ANKRA2, ANKRD13C, ANKRD44, ANKRD45, ANLN, ANXA9, AOC1, AP3S2, APEH, APOA2, APOL3, APOM, AQP2, ARCN1, ARHGAP29, ARHGEF19, ARL8A, ARMC2, ARRB1, ARSB, ASAP3, ASB3, ATAD3B, ATF3, ATP2B4, ATP5MC1, ATP6V1C1, ATP6V1D, ATPAF1, ATRAID, ATRIP, AUTS2, AZI2, B3GNT9, BAIAP2, BAIAP2L2, BAP1, BATE, BCAT1, BCKDHA, BCL2L13, BCL2L2, BLMH, BMPER, BOD1, BPIFA1, BSG, BSPRY, BTC, BTD, BTRC, C18orf54, C1QTNF12, C1QTNF4, C3orf38, C6orf223, C8orf76, C9orfl l6, CALCOCO1, CALHM1, CAT, CAVIN1, CAVIN4, CBWD1, CCDC113, CCDC121, CCDC14, CCDC174, CCDC86, CCDC93, CCIN, CCN2, CCN3, CCR6, CCSAP, CCT7, CD19, CD1B, CD36, CD46, CD86, CD96, CDC14A, CDH1, CDK2AP1, CDK7, CELA2B, CEP97, CFAP46, CFAP47, CHAMP1, CHMP2B, CHST8, CKB, CLEC11A, CLEC4M, CLEC9A, CLPB, CLPSL1, CLSTN1, CLUL1, CLYBL, CNDP2, CNTROB, COA6, COL15A1, COL21A1, COL22A1, COLQ, COMMD5, CORO6, COX11, CPA1, CPSF6, CPT2, CRB3, CREB3L2, CREM, CRHR2, CRTC2, CRYGD, CSK, CTBP2, CTNNA2, CTSS, CX3CR1, CYP20A1, CYP2C9, DADI, DAXX, DBF4, DCDC2, DCLK2, DCTN3, DCX, DDB1, DDHD1, DDO, DDOST, DDX21, DDX56, DEGS2, DEPDC7, DFFB, DIXDC1, DEK1, DNAI2, DNAJC16, DPPA4, DPYSE3, DPYSE4, DRG2, DUT, E2F7, EBNA1BP2, ECHDC1, ECHDC3, ECU, ECRG4, EDC3, EFCAB1, EFCAB12, EFCAB13, EFEMP2, EGR2, EIF2A, EIF4G3, EMP1, ENPP3, EPHA4, EPHB6, EPX, ERCC2, ERN2, ERV3-1, ESRP1, ETNK2, EVI2B, EXO1, EXTL3, EZH2, F10, F13B, F2, FAF2, FAM110A, FAM13C, FAM161B, FAM181A, FAM200A, FAM71A, FAM98A, FANCB, FATE1, FBXL13, FBXL16, FBXL20, FCRLB, FEM1C, FEV, FGF22, FGF3, FKBP14, FKBP5, FKBPL, FLOT1, FLVCR1, FLYWCH1, FMN1, FMO5, FOSB, FOXJ1, FOXS1, FPGT, FRMD5, FUT8, G6PC3, GAB3, GABRA5, GABRB2, GABRQ, GAD2, GALNT7, GAN, GAPDHS, GAPVD1, GBP4, GCNA, GDF6, GDF9, GDPD5, GET1, GET4, GGA1, GGA2, GGTLC1, GIMAP8, GKAP1, GLRB, GLT6D1, GLTPD2, GNAI2, GNL3, GORASP1, GORASP2, GOSR1, GPAA1, GPC5, GPR119, GPR37, GPR65, GPRASP2, GPRC5C, GPT2, GPX1, GRHL2, GRID1, GRINA, GSDME, GTF2H1, GTF2I, GTPBP2, GUSB, GYPA, H2BC4, HABP4, HACD3, HAGH, HAND2, HARS2, HAUS7, HAX1, HDAC9, HDHD3, HENMT1, HEPACAM2, HHEX, HIBCH, HID1, HK1, HLA-C, HLCS, HNF4A, HNRNPAB, HNRNPD, H0XA11, H0XD8, HP1BP3, HPS3, HPS4, HSDL1, HSP90B1, HTR1F, HTRA4, HVCN1, IBSP, IDS, IFNA8, IFNAR2, IFNGR1, IFNLR1, IGFBP1, IGKV1D- 17, IHO1, IK, IL10RA, IL13RA2, IL17RE, IL36G, IMPDH2, INF2, INKAI, INTS5, IP6K2, IPO5, IQCA1, IREB2, JAGN1, JMJD8, KAT14, KAT7, KAZALD1, KCNAB3, KCNG3, KCNJ14, KCNK12, KCNK4, KCNK9, KCNMB2, KCNV1, KCTD4, KHDRBS2, KHDRBS3, KIAA0930, KIF7, KIFC3, KLHL14, KLHL2, KLHL8, KREMEN2, KRT13, KRT79, LARS2, LCK, LECT2, LGALS12, LHX4, LIMA1, LMX1B, LNX1, LOXL1, LRP3, LRP5L, LRRC18, LRRC45, LRRC55, LRRC61, LRRN3, LTBR, LYVE1, MAFB, MAG, MAGT1, MANSC1, MAP1LC3C, MAP2K1, MAP2K4, MAP2K5, MAP6D1, MAPK14, MARVELD2, MCAM, MCF2L, MCM10, MCOLN3, MED1, MEM01, METTL14, MEX3B, MFAP4, MGAT4B, MGME1, MICU2, MIER3, MKS1, MLLT11, MLST8, MMP13, MOBP, MOVIO, MRPL3, MRPS27, MSTN, MTG2, MTHFD2, MTMR12, MTMR8, MYBL1, MYCL, MY01A, MYORG, NACC2, NADSYN1, NAE1, NBPF15, NCAM1, NCDN, NCLN, NDUFA2, NDUFB7, NECAP1, NECAP2, NECTIN1, NECTIN2, NEK3, NF2, NFIL3, NGFR, NIBAN1, NID2, NIF3L1, NIPSNAP3B, NKX2-8, NOA1, NOXI, NPLOC4, NPNT, NR1I2, NRM, NRTN, NT5DC1, NTPCR, NUP37, NXF3, NXPE3, NXT1, OAT, OIT3, OLFM2, OR1F1, OR1N1, OR52E8, OR9A4, 0RM2, OTUD7B, OTX1, OXCT2, P2RX4, P2RX5, P2RX7, P2RY6, P2RY8, P4HTM, PABPC3, PAF1, PAIP2, PARD6B, PARVA, PAX9, PCCA, PCDHGB1, PCLO, PDE1A, PDE4A, PDE9A, PDGFRA, PDHB, PDSS1, PDZD3, PDZD9, PELO, PFKL, PFKP, PI3, PI4K2A, PIGO, PIM1, PIMREG, PLA2G7, PLAGL1, PLAT, PLEKHO2, PLOD1, PLOD2, PLPP1, POFUT2, POGLUT2, POLR1C, POLR2L, POLR3C, POU3F2, PPA2, PPHLN1, PPP1R16B, PPP1R32, PPP1R9B, PPP2R2B, PPP2R3B, PRAME, PRDX3, PREB, PRELP, PRKCE, PRKCH, PRMT8, PRSS3, PSMB10, PSMC4, PSMD3, PTK6, PTPN18, PTPN2, PTPN9, PYROXD2, R3HDM2, RAB29, RAB30, RAB33B, RAB6B, RAB8B, RAD18, RAET1E, RAG2, RASGRP4, RASSF1, RBM12, RBM46, RCBTB2, RCC1L, RDX, RECQL4, REEP4, REG3A, RET, RGCC, RILP, RIMS2, RIN1, RINL, RITA1, RMND5A, RNF181, RPA3, RPL18, RPL28, RPS6KA2, RRM2, RRP15, RTN2, RUFY4, RUNDC1, SAMD13, SAMD4A, SCEL, SCGB2A2, SDCBP, SDHAF2, SEC23B, SEC63, SEMA3D, SEMA6D, SENP5, SEPTIN6, SEPTIN7, SERPINA10, SERPINE2, SFT2D2, SGCA, SGCB, SGIP1, SH3GL2, SHFL, SHOC2, SIAH2, SIGLEC7, SIRPG, SIRT6, SLC17A4, SLC22A5, SLC23A1, SLC23A3, SLC25A25, SLC25A3, SLC25A40, SLC25A41, SLC27A2, SLC27A3, SLC29A4, SLC2A8, SLC37A2, SLC39A12, SLC41A3, SLC44A5, SLC46A3, SLC51B, SLC6A5, SLCO4A1, SMAD3, SMG9, SMIM19, SMOX, SMPDL3B, SNF8, SNX5, SOCS6, SOD3, SOHLH2, SOX6, SOX8, SOX9, SPAG5, SPATA4, SPATCI, SPNS1, SPOCK1, SPRED1, SPSB1, SRF, SRM, SRPK2, SRRD, SSH3, SSRP1, ST6GALNAC6, ST7, ST8SIA3, STAT4, STIMATE, STING1, STOML2, STX1B, STX5, STXBP2, SYCE1, SYT11, SYT9, TAB2, TAMM41, TBC1D21, TBCD, TBL3, TBRG4, TBX10, TBX21, TBX22, TCF7L2, TERF2IP, TES, TEX10, TEX2, TEX45, TFAP2A, TFF2, TGFB3, THOC1, THOC3, THOP1, THRB, THRSP, THUMPD3, TIPRL, TLE1, TLE4, TMED3, TMEM107, TMEM183A, TMEM64, TMEM98, TMLHE, TMOD4, TNFSF15, TNNT2, TOMM22, TPRG1L, TPST2, TRAF3IP1, TRIM40, TRIM47, TRIP13, TRMT13, TRMT1L, TRMT2A, TRPV2, TRPV5, TSGA10, TSPAN16, TSPAN2, TSPAN32, TSPAN4, TSPAN5, TUBAL3, TUBB, TUBB3, TUBB4A, TUBGCP2, TULP4, TUT7, TXNIP, U2AF1L4, UBA6, UBASH3B, UBE2B, UBE2G1, UBE2I, UBE2O, UBQLN2, UBR2, UGDH, ULK3, UMOD, UNC13D, UQCRC2, UQCRFS1, USP10, USP28, USP33, UVRAG, VAX2, VEGFA, VNN1, VNN3, VPS72, VRTN, VSIG8, VTI1B, VWC2, WBP11, WDFY2, WDR4, WDR60, WDTC1, WEE1, WFDC13, WFDC6, WNT2B, WRAP73, WSB1, XKR6, XP07, XRCC5, XXYLT1, YAP1, YBX3, YY1AP1, ZBTB18, ZBTB39, ZDHHC20, ZDHHC5, ZIK1, ZKSCAN1, ZNF101, ZNF16, ZNF175, ZNF224, ZNF25, ZNF334, ZNF35O, ZNF462, ZNF485, ZNF519, ZNF653, ZNF677, ZNF71, ZNF761, ZNF776, ZNF785, ZNHIT2, and ZSCAN18.
65. The method of claim 63 or claim 64, wherein the immunosuppressive resistance gene is selected from the group consisting of COPZ2, GNL3, LIMA1, and LTBR.
66. The method of claim 61, wherein the immunosuppressive cellular environment comprises a TGF-P driven immunosuppressive cellular environment.
67. The method of claim 66, wherein the immunosuppressive resistance gene is selected from the group consisting of ABCE1, ABHD12B, ABLIM1, ABO, ACAA2, ACBD4, ACBD6, ACD, ACSBG1, ACSL5, ACTR1A, AD ARBI, ADCK2, ADGRG3, ADGRG7, ADPRHL1, AFAP1, AGGF1, AHI1, AIFM3, AKIRIN2, ALAD, ALDH1A1, ALK, AL0X15B, AMDHD2, AMY2B, ANGPT2, ANKMY1, ANKRD13D, ANKRD33B, AP3S2, APIP, APOA5, APOC4, APP, APPL2, AQP5, ARFGAP1, ARL11, ARMC12, ARRB2, ARSI, ART5, ATP6V0C, ATXN3, B3GAT3, BACE2, BAG1, BCAT1, BCKDHA, BCL7C, BIK, BIRC7, BPIFA2, C16orf70, C18orf32, Clorf35, Clorf87, C1QB, C1QL3, C1QTNF12, C3AR1, C7orf26, CACNG3, CACNG6, CALHM1, CALHM3, CAPN2, CAPZA2, CARD8, CASD1, CASK, CATSPER2, CAVIN3, CCDC106, CCDC121, CCDC153, CCDC68, CCDC70, CCDC93, CCL28, CCNL1, CCNY, CCT7, CD300C, CD300LD, CD96, CDC37, CDC42EP2, CDCA2, CDH13, CDH26, CDHR1, CDPF1, CDRT4, CEP120, CEP43, CERKL, CERS2, CES2, CFAP161, CFAP46, CGGBP1, CHID1, CHKA, CHRND, CIART, CITED2, CITED4, CLDN18, CLEC2D, CLSTN1, CLSTN3, CNDP2, COL6A2, COLCA2, COMT, COPZ2, CORO6, CPA6, CPT1C, CREB3L2, CRLF3, CRP, CSH1, CYP2C8, CYP2D6, CYP51A1, DBT, DCDC2, DCP2, DCUN1D5, DDB1, DDI2, DDX1, DDX53, DEAF1, DELEI, DEXI, DGKG, DHRS13, DKK4, DNAJB8, DNAJC11, DNTT, D0K5, DPP4, DPYSL4, DPYSL5, DSCC1, DUSP8, DYDC2, E2F2, EBP, ECU, EDN3, EGLN3, EIF1AY, EIF2AK1, ELAVL1, ELN, ELOA, ELOA2, EMC3, EMC4, ENKUR, EN0X1, EPB41L1, EPB41L4A, EPHX4, ERICH1, ESRP1, ETFDH, FAM161B, FAM207A, FAM20C, FAM76B, FAM78A, FBXL3, FBXO31, FBXO4, FCGR2A, FCN2, FCRL5, FCRLB, FGF18, FGFR1OP2, FGL1, FHL5, FIGNL1, FLU, FNDC9, FOXA3, FPGS, FREM1, FRMD8, FSD1L, FUZ, FXR1, GABBR2, GABRQ, GATAD1, GDF10, GDF5, GDF6, GGA1, GGT5, GID8, GJA10, GKAP1, GMCL1, GNA12, GNAI3, GNB5, GNL1, GNLY, GORASP1, GORASP2, GPC5, GPN2, GPR15, GPRASP2, GRIK2, GRINA, GRP, GSPT2, GTF2F1, GTPBP2, GXYLT1, H3C10, HAGH, HAPLN3, HCRTR1, HEPACAM2, HEXA, HIC2, HINT1, HLA-DOB, HLA-DQB2, HOXB9, HOXD3, HTR1A, HYLS1, IDS, IFNA10, IFNL3, IGFBP1, IGFBP4, IGHG1, IGHV7-81, IK, IL13RA1, IL17C, IL1R2, IL2RG, IMPA1, INKA2, INO80E, INPP5J, IQCG, IQUB, ITGB7, ITLN1, ITPA, JADE1, JAKMIP1, JAML, KCNAB2, KCND1, KCNMB1, KCTD10, KCTD13, KHDRBS3, KIAA0895, KIFC2, KLC2, KLHDC1, KLHL9, KLK10, KPTN, KRT79, KRTAP10-7, KRTAP4-4, L3MBTL4, LAMB3, LCN9, LDHA, LHX2, LIAS, LIMA1, LIMD1, LMNA, LMO2, LOXHD1, LPCAT2, LRFN5, LRP11, LRP3, LRRC18, LRRC45, LRRFIP2, LRTM1, LYPD1, LYPD5, LYPD6B, MACROD1, MACROH2A1, MAFF, MAGEB6, MAGEH1, MALT1, MANF, MAP6D1, MAPK15, MAPKAPK5, MAPRE3, MARCKS, MARS1, MAST2, MAST3, MAST4, MC3R, MCAM, MCM8, MCM9, MCU, MDGA2, METTL25, MGAT4B, MICU2, MIDN, MINDY1, MIPEP, MLST8, MMD, MMP11, MMUT, MORN1, MOXD1, MPND, MPP2, MPV17L2, MPZL1, MRAP2, MRM3, MRPL3, MRPL4, MRPL41, MRPL48, MRPL51, MRPS30, MRS2, MSMP, MSX1, MSX2, MTMR1, MTPAP, MUC3A, MVB12A, MX1, MXRA8, MYBL1, MYO1A, MYOC, MY0M3, NABP2, NAF1, NAGLU, NAIF1, NAXD, NCCRP1, NCDN, NDOR1, NDUFA7, NDUFS8, NELLI, NEUROG3, NFE2L3, NFIA, NFS1, NHLRC3, NINJ2, NIPAL3, NKAP, NMD3, NONO, NPDC1, NPNT, NR5A1, NRSN2, NTN5, NUBP1, NUDT3, NUDT9, NUP54, NUP62, NUP85, NXNL2, NXPE3, NYX, OAS1, ODF3, ODF3L2, OGGI, OLIG1, OLIG3, OPCML, OR10H1, OR4A15, OR4K17, OR51E1, OR5D14, OR8B12, ORC3, OSGIN1, OSTN, OTUB2, OTUD5, OXGR1, P2RY12, PACC1, PACSIN1, PACSIN2, PAK4, PARG, PARM1, PAX9, PCDH8, PCDHA10, PCDHB13, PCDHB2, PCSK7, PCYOX1L, PDE4A, PDE9A, PDHB, PDIA3, PDZD7, PEMT, PENK, PEX16, PFDN5, PFKFB3, PFKFB4, PFKL, PGRMC1, PHACTR3, PHF7, PICALM, PIGH, PIGP, PKM, PKP1, PLA2G3, PLAT, PLCG2, PLD6, PLEKHG5, PLEKHO2, PLP2, PLPPR2, PLXDC2, PMFBP1, PNMA2, PNOC, PNPT1, PGDN, POGLUT3, POLR3E, POLR3F, POU3F2, PPCDC, PPP1R12C, PPP2R1A, PPP2R2D, PPP3R2, PRDM1, PRKCD, PRLHR, PRMT2, PRNP, PRPF4, PRPF40A, PRR18, PRSS22, PSMA2, PSMD5, PTDSS1, PTGES2, PTK6, PTPN18, PTPRH, PTPRJ, PTPRS, PUM3, PWP1, PXMP4, PYROXD2, QPRT, RAB23, RAB39A, RAB42, RAB6B, RAD51B, RAFI, RALGPS1, RASSF2, RBM24, RBM3, RBM34, RBM46, RCC1L, RCN3, RDH10, RHBDD1, RHOH, RHOXF1, RILPL2, RIN1, RING1, RINL, RITA1, RNF126, RNF141, RNF144B, RNF220, RNF6, RO60, ROR1, RP2, RPH3AL, RPL30, RPL34, RPL6, RRS1, RSAD2, RTL8A, RUNDC1, RUNDC3A, RUSC1, RXFP3, SAFB2, SASS6, SBK1, SCG3, SCNN1A, SDF2L1, SDHAF2, SEC14L2, SEC61G, SEC63, SEL1L2, SEMA3G, SENP5, SERBP1, SERPINB5, SF1, SFRP1, SFT2D2, SFXN3, SGK1, SG01, SH3GL2, SH3GLB1, SHH, SHKBP1, SHOC2, SIVA1, SKIL, SLC12A1, SLC1A7, SLC22A13, SLC22A7, SLC25A20, SLC25A43, SLC27A6, SLC2A13, SLC2A8, SLC37A3, SLC45A2, SLC49A4, SLF1, SMAD6, SMARCE1, SMG5, SNAP91, SNAPC2, SNRPB, SNRPG, SNX14, SNX27, SOWAHA, SPAG5, SPATS2, SPIRE1, SPRTN, SPTLC2, SRC, SRF, SSH3, SSX3, ST3GAL6, STARD7, STIM1, STK3, STK35, STMN1, STOML3, STX10, STXBP4, SUSD3, SUSD6, SYP, TAC1, TAFA5, TBC1D19, TBCK, TBL3, TBRG4, TCAF1, TCEA2, TCF19, TCP11, TCP11L1, TDG, TDP2, TEX13A, TGM4, TGOLN2, THAP11, THBD, THOC5, TIMM29, TLR2, TM4SF4, TMED6, TMEM106B, TMEM178B, TMEM204, TMEM234, TMEM263, TMEM41A, TMEM86A, TMIGD2, TMPRSS11E, TMPRSS2, TNFAIP1, TNFAIP8L1, TNFRSF10C, TNFSF13B, TOR1B, TOX2, TPD52L2, TPM4, TPO, TPSD1, TPT1, TRAPPC10, TRAPPC8, TRIM40, TRIM55, TRIR, TRMT12, TRPC5, TRPV2, TSKS, TTC12, TTLL7, TUBA1C, TUBGCP2, TXNDC5, UBAC1, UBXN2A, UGT3A1, UIMC1, UNCI 19, UQCRFS1, USH1C, USP15, USP21, VAC14, VEGFA, VPS37B, VPS45, VRTN, WAPL, WDR1, WDR24, WDR54, WDR5B, WDR60, WDR61, WIPI2, WNT2, XAF1, YBX2, ZAP70, ZBTB5, ZBTB46, ZC2HC1A, ZC3H3, ZCCHC8, ZDHHC1, ZDHHC13, ZFP2, ZFP36L2, ZGRF1, ZMPSTE24, ZNF175, ZNF19, ZNF205, ZNF274, ZNF428, ZNF436, ZNF502, ZNF558, ZNF624, ZNF668, ZNF71, ZNF710, ZNHIT2, and ZSWIM1.
68. The method of claim 66 or claim 67, wherein the immunosuppressive resistance gene is ZBTB46.
69. The method of claim 61, wherein the immunosuppressive cellular environment comprises a regulatory T cell driven immunosuppressive cellular environment.
70. The method of claim 69, wherein the immunosuppressive resistance gene is selected from the group consisting of FOSB, AARSD1, ABCC10, ABHD5, ABI1, ACAD10, ACAD9, ACBD3, ACSL4, ACTB, ACTL7B, ADAT1, ADGRE5, ADIPOR2, ADORA3, AEN, AFP, AGFG2, AGPAT2, AHNAK, AHSA1, AIPL1, ALAS2, ALG3, ALK, AMDHD1, ANG, ANKRD1, ANKRD45, ANKRD54, ANLN, ANXA13, ANXA4, AOC1, APH1A, APRT, AQP1, ARAP1, ARC, ARFGAP1, ARHGAP12, ARHGAP25, ARHGEF7, ARID3B, ARMC7, ARNT2, ARPC1B, ARRDC4, ARSK, ASIP, ASS1, ATG4A, ATG9A, ATP23, ATP5F1B, ATP5MC2, ATP6V0C, ATP6V0D1, B9D2, BAB AMI, BAG5, BCHE, BLK, BMPR1B, BPIFC, BRF2, BSND, BTNL3, BYSL, C10orf82, C18orf25, C18orf54, Clorfll5, Clorf43, Clorf56, C1QTNF2, C1R, C2CD2, C5orfl5, C6orfl20, CAB, CA5B, CA8, CA9, CABS1, CALC0C01, CALCR, CALHM3, CAMK2A, CAMLG, CARD8, CASP1, CASP7, CASTOR1, CBX7, CCDC110, CCDC69, CCDC82, CCDC84, CCL2, CCL21, CCR8, CCSER1, CD151, CD300LF, CD48, CD83, CD86, CDH7, CDK1, CDPF1, CDR2, CELF1, CEP63, CERS1, CES3, CFAP410, CGGBP1, CHAF1B, CHMP2A, CHMP7, CHRM1, CHST9, CISH, CLC, CLDN6, CLEC5A, CLIC5, CLP1, CLTRN, CLUAP1, CMTM7, CNTF, COG3, COL25A1, COL8A2, C0MMD4, COPZ1, COPZ2, COQ4, COX6B2, CPLX2, CRACR2B, CROT, CRTAC1, CRY2, CSF1, CSNK1G2, CST9, CSTF3, CTDP1, CTNNA2, CTSF, CXCR6, CXXC1, CYP27A1, CYP2D6, CYTL1, DAXX, DBN1, DDHD2, DDX24, DES, DEXI, DGLUCY, DHX36, DNAI2, DNAJA2, DNAJB5, DNAJB6, DNAJC11, DNAJC27, DNAJC6, DOK5, DOK6, DRGX, DUS1L, DUSP5, DZIP1L, ECI2, EFCAB7, EGFL6, EGLN3, EIF3K, EIF4EBP1, ELSPBP1, EMC3, EPDR1, EPN1, EPO, ERFE, ERVK3-1, ESMI, F2RL2, FAAP100, FADS1, FAM172A, FAM53C, FAM71C, FAM81A, FBLN5, FBXL16, FBXO7, FBXW11, FCGR3A, FCRL5, FDFT1, FETUB, FEZF2, FGF10, FGF19, FGL1, FKBP5, FKBP9, FMOD, FNDC9, FRMD3, FRMD5, FRZB, FUT3, GAB3, GABRA4, GABRG2, GALNT2, GALNTL6, GAS7, GBA2, GBP6, GEMIN8, GET1, GFM2, GFRA3, GK2, GLIPR1, GLRA2, GLRB, GLYCTK, GNA14, GNB3, GNL3, GOLM2, GPATCH2L, GPATCH3, GPM6B, GPR176, GPR45, GRAMD1B, GRB7, GRK2, GRK7, GSTM3, GXYLT1, GZMM, HAPLN3, HAUS2, HBG2, HCRTR1, HDAC8, HDGF, HEMK1, HERPUD1, HESX1, HEXA, HMHB1, HOXB5, HOXB6, HOXD3, HOXD4, HPGDS, HSD17B6, HSD17B8, HSPA2, HTN1, HTR5A, IFITM3, IFNA10, IGF1, IGFALS, IGHM, IL11RA, IL17A, IL17RE, IL2RB, IL12RB2, IL4, INSL6, ISL2, ISM2, IST1, ITPRID2, JAML, JUN, JUNB, KBTBD7, KCNA6, KCNN3, KEAP1, KIF3A, KIFC2, KIR2DL1, KLHDC2, KLHDC7B, KLHL9, KRT19, KRT6A, LARS2, LCK, LCN1, LDAH, LDLRAD4, LETMD1, LHX4, LHX9, LIN28A, LIPG, LNX1, LPAR5, LPL, LRFN3, LRRC15, LRRC2, LRRC34, LRRC42, LTBR, LYZ, MAF1, MAGOH, MAN1A1, MAN1B1, MAP2K6, MAP3K7, MAP4K5, MAPKAPK2, MAPKAPK5, MARCHF1, MARCHF2, MBNL1, MBP, MC3R, MCAM, MED26, MEIS3, METTL27, METTL2B, MIA2, MIF, MIPOL1, MLKL, MMD, MMP10, MOB4, MPHOSPH8, MPND, MPZL1, MR1, MRAS, MRM3, MRPL21, MRPS24, MRPS28, MS4A3, MS4A5, MSRA, MTA2, MTA3, MTHFD2, MTMR3, MTPAP, MTRF1L, MUS81, MYCL, MYCN, MYL10, MYL9, MYOC, MY0M3, MYORG, NAA80, NAPSA, NARF, NAT1, NCK1, NDE1, NDOR1, NDUFA13, NDUFA4, NEK5, NELFE, NFIB, NFKBIB, NINJ2, NIPAL1, NKAIN2, NKAP, NMB, NOC4L, NPIPB15, NRARP, NRSN2, NTF3, NXNL2, OBP2A, OLR1, OR10AG1, OR10K2, OR14C36, OR1F1, OR2M3, OR2T8, OR5C1, OR7A5, OR9Q1, ORAI3, ORC2, 0RM1, 0RM2, OSGIN1, OSR2, OTUD5, P3H4, P4HA3, P4HTM, PAK2, PAK4, PBX2, PCDHA2, PCDHB12, PCDHGA2, PDYN, PFDN4, PFKP, PFN4, PGK1, PGLYRP1, PHF23, PHF7, PHKG1, PHOSPHO1, PI15, PI3, PI4KB, PITHD1, PKIA, PKNOX2, PLA2G3, PLA2G7, PLAUR, PLEKHA8, PLEKHO2, PLET1, PLOD2, PNPT1, POPDC3, PPM1D, PPME1, PPP1R2, PPP1R32, PPP2R2B, PPP2R3C, PPP2R5C, PPP6R2, PRKCB, PRKRIP1, PRKY, PRMT8, PRSS3, PRUNE2, PSCA, PSG1, PTGER3, PTK6, PTOV1, PTPRO, PTTG1IP, PUDP, PWWP3B, PYCARD, PYGB, QPCT, QPRT, RABI IB, RAB25, RAB28, RAB34, RAB40B, RABEP2, RADU, RAET1E, RAMP1, RARS1, RAX2, RBBP5, RCHY1, RCN3, REEP2, RFC4, RFPL2, RFX3, RIBCI, RINL, RIPK4, RLBP1, RNASE9, RNASET2, RNF111, RNF112, RNF144B, RNF24, RNF38, RNF7, ROPN1L, RP9, RPL6, RPS2, RPS3A, RPS6, RRAGA, RRAGD, RRP1, RRP9, RSAD1, RUBCN, RUNX1T1, RUVBL1, SAMHD1, SARS1, SCNN1A, SCNN1B, SCNN1G, SEL1L2, SELENBP1, SEPHS1, SEPTIN10, SEPTIN12, SERINC2, SERPINA3, SERPIND1, SERPINE1, SERPINE3, SETD3, SFRP4, SGK2, SH3KBP1, SHARPIN, SHISA3, SHOC2, SHOX, SIGLEC7, SIRPB2, SIRPG, SIRT3, SKIL, SLC13A1, SLC14A1, SLC20A2, SLC22A13, SLC22A31, SLC22A8, SLC25A1, SLC25A46, SLC25A47, SLC25A48, SLC2A8, SLC37A3, SLC39A7, SLC5A12, SLC6A19, SLC6A7, SLC7A9, SLCO1A2, SLFNL1, SMG9, SMPX, SNORC, SNRNP25, SNRPB, SNRPN, SNX16, SOAT1, SOCS5, SPATA22, SPATS2, SPC25, SPG21, SPINT1, SPINT2, SPNS2, SPP2, SQOR, SRC, SRFBP1, SRP54, SRP9, SRSF9, SSBP2, SSPN, STAP1, STARD7, STIM1, STK11, STX8, SULT4A1, SUMF2, SURF6, SYMPK, SYNCRIP, SZT2, TAS2R40, TAS2R60, TBCC, TBRG4, TBX20, TBX3, TON, TCEA1, TCEA2, TCF19, TCF7L2, TCN2, TCTN1, TDP2, TENT5C, TEX35, TFCP2L1, TFDP2, TGFB3, THAP12, TIAM2, TICAM2, TIPIN, TKT, TLE4, TM2D2, TM9SF3, TMEM106B, TMEM143, TMEM160, TMEM211, TMEM237, TMEM270, TMEM30A, TMEM39B, TMEM45A, TMEM68, TMPRSS3, TNFSF12, TOMM70, TOR1AIP1, TOX, TPP1, TPRKB, TPST2, TRAPPC12, TRDMT1, TRIM10, TRIM47, TRIM63, TRIP10, TRMO, TRMT44, TRPV4, TSN, TSPAN31, TSPEAR, TSSK3, TTBK2, TTC32, TUBA3C, TUBA3D, TUT7, TYW3, UBA1, UBASH3B, UBE2S, UBXN2A, UCHL3, UCHL5, UQCR10, USP1, USP19, VAT1L, VMA21, VPS29, VPS36, VSTM2A, VWA1, WAS, WDFY1, WDR4, WDR59, WDR63, WDR78, WNT11, WNT3A, WNT9A, YAF2, YJU2, ZBTB48, ZC3HAV1L, ZCCHC2, ZCCHC7, ZFP36L2, ZIC3, ZNF165, ZNF830, ZP2, ZSCAN21, and ZSCAN9.
71. The method of claim 69 or claim 70, wherein the immunosuppressive resistance gene is selected from the group consisting of FOSB, IL12RB2, LTBR, MCAM, PTK6, and SKIL.
72. The method of claim 61, wherein the immunosuppressive cellular environment comprises a macrophage driven immunosuppressive cellular environment.
73. The method of claim 72, wherein the immunosuppressive resistance gene is selected from the group consisting of AB AT, ABHD12, ABI1, ACP7, ACSM3, ACTA2, ADRB3, AGAP1, AGTPBP1, AIF1L, AIFM1, AIM2, AK7, ALG1, ALG3, ALKBH1, ALKBH5, ALOXE3, ALPP, AMELX, AMTN, ANKH, ANKRD22, ANKRD39, ANKRD9, ANKS4B, ANOS1, ANXA8, ANXA9, AP3M1, AP3S1, APEX2, AQP1, AQP9, ARAF, ARFGAP3, ARHGAP20, ARHGEF1, ARL4D, ARMC2, ARNTL, ASAP3, ASB3, ASIC2, ASPH, ASTE1, ATAD3A, ATG3, ATG4C, ATP5F1B, ATP6V1B2, AUTS2, AVP, AVPR1A, B3GALNT2, B3GALT4, B3GNT2, BAB AMI, BAP1, BCAP31, BCCIP, BCL2L2, BCR, BDNF, BECN1, BEND2, BEND7, BLOC1S4, BMP5, BMPR1B, BRD3, BRF2, BRINP3, BTBD17, BTNL3, C10orf62, C12orf42, C14orf28, Clorf210, C2orf78, C6, C7orf31, CAB, CACNB3, CALR3, CARD14, CARD19, CASC1, CASP10, CASQ2, CBFB, CBX3, CBX4, CCDC141, CCDC148, CCDC68, CCDC8, CCDC82, CCIN, CCNG2, CCNY, CCR1, CCR10, CCR3, CD19, CD244, CD47, CD5L, CD83, CDC42EP1, CDC42EP4, CDCA2, CDCP2, CDH13, CDYL2, CEP120, CEP63, CERCAM, CERKL, CFAP100, CFAP20, CFAP410, CFAP47, CFAP91, CFHR1, CHGA, CHUK, CIRBP, CLCNKB, CLDND1, CLU, CMTR1, CNPPD1, COA3, COG1, COL22A1, COL25A1, COL6A2, COL8A2, COPB1, COPS3, COQ3, COQ8B, COX18, CPOX, CPQ, CPT1A, CPXM1, CRACR2A, CREB3L1, CRNN, CRTAP, CRYGD, CS, CSF1, CSF1R, CSF2RB, CSF3R, CSGALNACT1, CSTF2, CTCF, CTSV, CUEDC1, CUL2, CXADR, CYB5R2, CYP20A1, CYP26A1, CYP2C19, CYP8B1, DAAM2, DAB1, DACT2, DARS1, DBN1, DCAF4L2, DCAF8, DCT, DDO, DDR1, DDX43, DDX54, DEAF1, DECR1, DENR, DESI2, DHX40, DIP2A, DKK4, DNAAF3, DNAI2, DNAJC7, DNAL1, DPP4, DPT, DPYSL4, DRG1, DSCAM, DTL, DTX2, DUS1L, DUSP12, E2F7, ECHI, EFEMP2, EFHC1, EFS, EIF2B3, EIF4A2, EIF5, ELAVL4, ELL, ELMOD3, ELN, ELOA, ELOA2, ELP3, EMC7, ENCI, ENDOV, ENPP3, ENTPD5, E0LA2, EPB41L1, EPHB6, EPN1, ERCC6, ESRP1, ESRRA, ETFDH, EX01, EXOC3, EXOSCIO, EXOSC8, EYS, F3, FABP6, FAM117A, FAM117B, FAM118B, FAM161B, FAM200A, FAM20A, FAM47A, FARSA, FBLN1, FBLN5, FBP1, FBXO24, FBXO40, FCHO1, FCRL5, FCRLB, FECH, FEM1B, FGF3, FGG, FKBP6, FLVCR1, FMNL1, FMO5, FNIP1, FOS, FOSB, FOXD4, FOXJ1, F0XM1, FOXN3, FOXRED2, FREM1, FRMD5, FRS2, FSTL1, FSTL4, FSTL5, FTMT, FXR2, G6PD, GABPB1, GABRB1, GALNT7, GANAB, GAPDHS, GATA2, GBP4, GCAT, GCGR, GCNT7, GCSAML, GDF2, GDF6, GET4, GGA1, GHDC, GINS4, GK, GKAP1, GLB1, GLRA2, GLRX5, GLYR1, GMCL2, GNA14, GNAT2, GNB3, GNL1, GNL3, GPAT4, GPC3, GPC4, GPC5, GPR84, GRHL3, GRHPR, GRIA4, GRID1, GRIK3, GRK4, GRM3, GRM8, GRN, GSDME, GSK3A, GSN, GTF2A1, GTF2B, GTF2E1, GUCY1B1, GYSI, HABP2, HADHA, HADHB, HDAC10, HEPHL1, HERPUD2, HES1, HEXD, HHIPL2, HINT2, HLA-C, H0MER3, HOOK3, HOXB6, HOXD3, HPS3, HPS5, HSD11B2, HSD17B13, HSD17B7, HSD3B1, HSPB9, HTR2B, IFNA6, IFNL1, IFT27, IGHA1, IGSF10, IGSF21, IL10RB, IL12RB2, IL13RA1, IL21, IL26, IL7R, ILVBL, IMPG1, INA, INHA, INSL4, INTU, IQUB, IRAG1, IRAKI, IREB2, IRF3, IRF5, IRX3, ITFG1, ITGB2, ITGB7, ITIH5, ITM2B, JAKMIP1, KANSL3, KAT5, KCNG3, KCNJ14, KCNK12, KCNK2, KCNK9, KCNMB3, KCTD12, KCTD4, KIAA2013, KIF2C, KIF3A, KIF3B, KIFC2, KITLG, KLC3, KLHDC2, KLHL13, KLHL21, KLHL8, KLK1, KRT6A, KXD1, L3MBTL4, LAD1, LAMP1, LAP3, LARS2, LDLRAD3, LGMN, LHX2, LHX4, LIMA1, LIMD1, LIPH, LIPT1, LKAAEAR1, LNPEP, LOXHD1, LOXL3, LTA4H, LTBR, LUC7L2, LYL1, LZTS2, MAG, MAN1A1, MANEA, MANF, MAP3K7, MAP4K5, MAP6D1, MAPK15, MAPK8, MAPKAPK5, MAPKBP1, MARS2, MATN2, MBLAC1, MCAM, MCOLN2, MCOLN3, MDH1B, MDM4, MEAK7, MECP2, MED1, ME0X1, METAP1, MFAP3L, MFNG, MITD1, MLEC, MLH1, MLLT3, MMP10, MMP16, MMS19, MNAT1, MON1B, MPP2, MPP7, MRM3, MRPL19, MRPL3, MRPL47, MSLN, MSRA, MTARC2, MTHFR, MTMR4, MTMR6, MUTYH, MVP, MYBL1, MYCN, MY01A, MYO5C, MYOC, MY0M3, MYORG, MYT1, NAGLU, NAMPT, NCKIPSD, NDC80, NDRG4, NDUFAF7, NDUFB6, NDUFS8, NDUFV2, NECTIN4, NELFA, NFIB, NIF3L1, NIPAL1, NIPBL, NMD3, NOC2L, NPLOC4, NPY, NPY2R, NQO1, NR2E1, NR6A1, NT5DC2, NTM, NTN5, NTNG1, NUDT8, NUP210, NXPE3, OAS1, OAS2, ODF2, OGA, OGFRL1, OLFM2, OLFML1, OLFML2B, OLIG1, OLR1, OMP, OR4D1, OR4S2, OR52N5, OR52W1, OR5B3, OR9Q1, ORMDL2, OSBPLIO, OSGEPL1, OTX1, OXSR1, P2RX6, P2RY1, PACS2, PAEP, PAFAH1B2, PAK4, PARL, PARP9, PARS2, PC, PCDHA1, PCDHA6, PCDHGA2, PCDHGA5, PCSK2, PDCD6, PDE4A, PDIA3, PDK2, PDZD9, PENK, PFKL, PGAM5, PGK1, PGM2, PHF11, PHYH, PICALM, PIK3R1, PIP5K1B, PLAGL1, PLCD4, PLD1, PLEK, PLEKHG5, PLEKHS1, PLIN1, PLK1, PLPPR2, PM20D1, PNMA8A, POLE, POLI, POLR2K, POLR3F, POU3F2, PPA2, PPIG, PPM1D, PPP1R12C, PPP1R16B, PPP1R32, PPP2R3C, PPP2R5C, PPP2R5D, PRAC1, PRAM1, PRAME, PRDM1, PRDX3, PRKAA2, PRKAG2, PRKAR1B, PRKCE, PRMT1, PROC, PRRT2, PRSS45P, PRSS48, PSAPL1, PSD3, PSEN1, PSMC4, PSMD14, PTBP3, PTCD2, PTDSS1, PTGER2, PTK6, PTPN12, PTPRN, PUF60, PUM1, PYDC1, PYGB, RAB1A, RAB33B, RAB42, RABL6, RAD18, RAD51, RAET1G, RALBP1, RASSF4, RBBP7, RBM12, RBM38, RBM4, RBM46, RBM4B, RBX1, RCBTB2, RCC1L, RDH10, RDH12, REC8, REN, RET, RFC2, RFC5, RFX4, RGS16, RHAG, RHEX, RHOBTB2, RICTOR, RIMBP2, RIMS3, RIOK3, RIPK1, RIPK2, RIT1, RMDN3, RNASEH2B, RNF114, RNF148, RNF213, RNF6, RNPEPL1, RO60, RORA, RPL30, RPS14, RPS4X, RPS6KA2, RPS6KB1, RSRC1, RTF1, RTN1, RUBCN, RUNDC1, RUNX3, S100PBP, SAMHD1, SCAMP2, SCIN, SCNN1D, SCRN2, SDSL, SEC63, SEL1L3, SELL, SENP3, SEPTIN10, SEPTIN8, SERINC3, SERPINF1, SGK3, SGMS1, SH3GLB1, SHOC2, SIAH2, SIGLEC10, SIGLEC12, SIGLEC7, SIRPG, SIRT5, SKAP1, SKIL, SLC15A3, SLC16A1, SLC16A7, SLC18A2, SLC1A7, SLC20A2, SLC22A2, SLC22A23, SLC22A24, SLC23A2, SLC25A19, SLC25A25, SLC25A47, SLC26A2, SLC2A4, SLC2A8, SLC36A3, SLC37A2, SLC37A3, SLC39A14, SLC43A1, SLC5A11, SLC5A12, SLC5A7, SLC6A7, SLC7A1, SLCO1A2, SLITRK3, SMARCD3, SMOX, SNCAIP, SNTA1, S0CS6, SOX9, SPATA2, SPCS3, SPN, SPNS2, SPOCK3, SPOUT1, SPRR4, SRC, SRD5A3, SRFBP1, SRMS, SSMEM1, SSX2IP, ST7L, STEAP1, STIM1, STK11, STK17A, STK17B, STK3, STK39, STXBP1, STXBP2, STXBP3, STXBP5, SUGCT, SUN5, SUSD2, SYNCRIP, TAB2, TBC1D10A, TBC1D22B, TBC1D9B, TBCD, TBL1X, TBX6, TON, TCF25, TCTN2, TDGF1, TEAD2, TESK1, TESK2, TEX48, TFAP2A, TFAP4, TGFB1, TGFBI, TGOLN2, THAP3, THAP7, THEM4, THOC1, THOC7, THOP1, THSD4, TIAM2, TICAM2, TLE6, TLK1, TM9SF4, TMED2, TMEM259, TMEM62, TMIE, TMLHE, TMOD1, TMTC3, TNF, TNS1, TOMM70, TRAF3IP1, TRAM1, TRAP1, TRAPPC3, TRAPPC4, TRAPPC8, TRAPPC9, TRIB3, TRIM32, TRMT13, TRPM3, TRPV4, TSGA10, TSPEAR, TSPYL6, TTC13, TTC16, TTC26, TTC38, TTC8, TTF2, TTLL2, TTLL7, TUBA3E, TUBA8, TUBB3, TUBB4B, TUFM, TYMS, UBE2O, UBE2Z, UBE3A, UBE3C, UB0X5, UGDH, UGP2, UGT3A1, UGT8, UMOD, USP1, USP49, UXS1, VANGL2, VASN, VEGFA, VIL1, VPS45, VWA1, WAPL, WDR37, WDR63, WDR90, WDR91, WNT4, WWOX, XRCC5, YBX2, YME1L1, ZBTB14, ZBTB18, ZBTB46, ZC3H10, ZC3H11A, ZCCHC8, ZDHHC11, ZDHHC15, ZDHHC6, ZFYVE21, ZGRF1, ZMAT3, ZNF165, ZNF18, ZNF19, ZNF20, ZNF224, ZNF25, ZNF277, ZNF334, ZNF350, ZNF354C, ZNF396, ZNF398, ZNF436, ZNF461, ZNF467, ZNF496, ZNF560, ZNF565, ZNF566, ZNF597, ZNF610, ZNF623, ZNF668, ZNF74, ZP1, ZPLD1, ZSCAN25, and ZSWIM2.
74. The method of claim 72 or claim 73, wherein the immunosuppressive resistance gene is selected from the group consisting of CD47, CD86, FOSB, GSDME, IL12RB2, IL26, LIMA1, LTBR, PTK6, SIRPG, SKIL, SRC, STK11, YBX2, and ZTBTB46.
75. The method of any one of claims 61-67 and 69-74, wherein the immunosuppressive resistance gene is LTBR.
76. A method of modifying a population of lymphocytes to overexpress an immunosuppressive resistance gene comprising the steps of:
(i) collecting peripheral blood mononuclear cells (PBMCs) from an individual,
(ii) isolating lymphocytes comprising CD8+ T cells, CD4+ T cells, naive CD4+ T cells, or regulatory T cells from the PBMCs of step (i),
(iii) culturing, activating, and/or differentiating the lymphocytes in T-cell media, and
(iv) transducing the lymphocytes with a vector encoding the immunosuppressive resistance gene, wherein the transduced lymphocytes overexpress the immunosuppressive resistance gene.
77. The method of claim 76, wherein the exogenous nucleic acid further comprises a chimeric antigen receptor (CAR).
78. The method of claim 76 or claim 77, wherein the exogenous nucleic acid further comprises a T cell receptor (TCR).
79. The method of any one of claims 76-78, wherein the peripheral blood mononuclear cells are obtained from leukapheresis.
80. The method of any one of claims 76-79, wherein the CD8+ and CD4+ T cells are isolated sequentially.
81. The method of any one of claims 76-80, wherein the naive CD4+ T cells are differentiated into activated CD4+ T cells.
82. The method of any one of claims 76-81, wherein the naive CD4+ T cells, CD8+ T cells, and CD4+ T cells are activated with Immunocult Human CD3/CD28 T-cell Activator (Stemcell).
83. The method of any one of claims 76-82, wherein the naive CD4+ T cells are differentiated into induced regulatory T cells with TGF-pi and retinoic acid.
84. The method of any one of claims 76-83, wherein the lymphocytes are transduced with a lentivirus, an adenovirus, a retrovirus, a baculovirus, a genome editing nuclease, or a transposable element.
85. The method of any one of claims 76-84, wherein the PBMCs collected from the individual are cryopreserved within 24-48 hours of collection.
86. The method of any one of claims 76-85, wherein the transduced lymphocytes are enriched by positive selection.
87. The method of claim 86, wherein the positive selection comprises culturing the transduced lymphocytes in cell culture media supplemented with puromycin.
88. A method of identifying a gene that confers resistance to an immunosuppressive cellular environment of a modified lymphocyte when expressed in the modified lymphocyte, the method comprising:
(i) obtaining a lymphocyte population comprising a mixture of CD4+ and CD8+ cells obtained from the same individual,
(ii) transducing the lymphocyte population with a plurality of viral vectors, each viral vector encoding a gene linked to one or more barcodes,
(iii) transiently stimulating the transduced lymphocytes,
(iv) exposing the transduced lymphocytes to an immunosuppressive environment,
(iv) isolating a transduced lymphocyte from the lymphocyte population of (iv), and
(v) detecting the presence of the gene and/or the linked barcodes in the isolated lymphocyte; wherein the detected gene is effective to confer resistance to the immunosuppressive environment of the modified lymphocyte that expresses the gene.
89. The method of claim 88, wherein the immunosuppressive cellular environment is selected from the group consisting of adenosine immunosuppression, TGF-P immunosuppression, Treg immunosuppression, and macrophage immunosuppression.
PCT/US2025/017337 2024-02-27 2025-02-26 Methods and compositions for improving t cell function in an immunosuppressive environment Pending WO2025184170A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463558573P 2024-02-27 2024-02-27
US63/558,573 2024-02-27

Publications (1)

Publication Number Publication Date
WO2025184170A1 true WO2025184170A1 (en) 2025-09-04

Family

ID=94968938

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/017337 Pending WO2025184170A1 (en) 2024-02-27 2025-02-26 Methods and compositions for improving t cell function in an immunosuppressive environment

Country Status (1)

Country Link
WO (1) WO2025184170A1 (en)

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010053572A2 (en) 2008-11-07 2010-05-14 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
WO2012170930A1 (en) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc Lipid nanoparticle compositions and methods for mrna delivery
WO2013182683A1 (en) 2012-06-08 2013-12-12 Ethris Gmbh Pulmonary delivery of messenger rna
WO2015090230A1 (en) 2013-12-19 2015-06-25 Novartis Ag Human mesothelin chimeric antigen receptors and uses thereof
WO2016138846A1 (en) * 2015-03-02 2016-09-09 Shanghai Sidansai Biotechnology Co., Ltd Reducing immune tolerance induced by pd‐l1
WO2017079694A2 (en) 2015-11-04 2017-05-11 Priceman Saul J Chimeric antigen receptors targeting her2
WO2019118902A2 (en) * 2017-12-15 2019-06-20 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for inhibiting t cell exhaustion
US10561686B2 (en) 2018-01-12 2020-02-18 Innovative Cellular Therapeutics CO., LTD. Modified cell expansion and uses thereof
US20220184119A1 (en) 2019-02-08 2022-06-16 Biontech Cell & Gene Therapies Gmbh Chimeric antigen receptor-modified cells for the treatment of cldn6-expressing cancers
WO2022182890A1 (en) * 2021-02-25 2022-09-01 Lyell Immunopharma, Inc. Codon-optimized nucleotide sequences encoding an ap-1 transcription factor
US20220306711A1 (en) 2014-04-01 2022-09-29 Biontech Cell & Gene Therapies Gmbh Claudin-6-specific immunoreceptors and t cell epitopes
US20220339193A1 (en) 2018-11-20 2022-10-27 Innovative Cellular Therapeutics Holdings, Ltd. Modified Cell Expressing Therapeutic Agent and Uses thereof
WO2023279049A1 (en) * 2021-06-30 2023-01-05 New York Genome Center, Inc. Methods and compositions for improved immunotherapies
US20230220343A1 (en) 2014-05-15 2023-07-13 National University Of Singapore Immune Cells Expressing Membrane-Bound Interleukin 15 (mbIL15) and Uses Thereof
WO2023172514A1 (en) 2022-03-07 2023-09-14 Catamaran Bio, Inc. Engineered immune cell therapeutics targeted to her2 and methods of use thereof

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010053572A2 (en) 2008-11-07 2010-05-14 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
WO2012170930A1 (en) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc Lipid nanoparticle compositions and methods for mrna delivery
WO2013182683A1 (en) 2012-06-08 2013-12-12 Ethris Gmbh Pulmonary delivery of messenger rna
WO2015090230A1 (en) 2013-12-19 2015-06-25 Novartis Ag Human mesothelin chimeric antigen receptors and uses thereof
US20220306711A1 (en) 2014-04-01 2022-09-29 Biontech Cell & Gene Therapies Gmbh Claudin-6-specific immunoreceptors and t cell epitopes
US20230220343A1 (en) 2014-05-15 2023-07-13 National University Of Singapore Immune Cells Expressing Membrane-Bound Interleukin 15 (mbIL15) and Uses Thereof
WO2016138846A1 (en) * 2015-03-02 2016-09-09 Shanghai Sidansai Biotechnology Co., Ltd Reducing immune tolerance induced by pd‐l1
WO2017079694A2 (en) 2015-11-04 2017-05-11 Priceman Saul J Chimeric antigen receptors targeting her2
WO2019118902A2 (en) * 2017-12-15 2019-06-20 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for inhibiting t cell exhaustion
US10561686B2 (en) 2018-01-12 2020-02-18 Innovative Cellular Therapeutics CO., LTD. Modified cell expansion and uses thereof
US20220339193A1 (en) 2018-11-20 2022-10-27 Innovative Cellular Therapeutics Holdings, Ltd. Modified Cell Expressing Therapeutic Agent and Uses thereof
US20220184119A1 (en) 2019-02-08 2022-06-16 Biontech Cell & Gene Therapies Gmbh Chimeric antigen receptor-modified cells for the treatment of cldn6-expressing cancers
US20220307039A1 (en) 2021-02-25 2022-09-29 Lyell Immunopharma, Inc. Codon-optimized nucleotide sequences encoding an ap-1 transcription factor
WO2022182890A1 (en) * 2021-02-25 2022-09-01 Lyell Immunopharma, Inc. Codon-optimized nucleotide sequences encoding an ap-1 transcription factor
WO2023279049A1 (en) * 2021-06-30 2023-01-05 New York Genome Center, Inc. Methods and compositions for improved immunotherapies
WO2023172514A1 (en) 2022-03-07 2023-09-14 Catamaran Bio, Inc. Engineered immune cell therapeutics targeted to her2 and methods of use thereof

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
DATBNGER ET AL., NAT METHODS, vol. 14, no. 3, March 2017 (2017-03-01), pages 297 - 301
DUAN HONGXIA ET AL: "CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 131, no. 21, 7 September 2021 (2021-09-07), US, pages 1 - 17, XP093043067, ISSN: 1558-8238, DOI: 10.1172/JCI148568 *
FEUCHT ET AL., NAT. MED., vol. 25, no. 1, 2018, pages 82 - 88
GOEDDEL: "Gene Expression Technology: Methods in Enzymology", vol. 185, 1990, ACADEMIC PRESS
HAQUE ET AL., GENOME MEDICINE, vol. 9, 2017
HARADHVALANICHOLAS JET, NATURE MEDICINE, vol. 28, no. 9, 2022, pages 1860 - 1871
HWANG ET AL., EXP MOL MED, vol. 50, no. 8, 7 August 2018 (2018-08-07), pages 96
JAITIN ET AL., CELL, vol. 167, no. 7, 15 December 2016 (2016-12-15), pages 1883 - 1896
LEGUT MATEUSZ ET AL: "A genome-scale screen for synthetic drivers of T cell proliferation", NATURE,, vol. 603, no. 7902, 16 March 2022 (2022-03-16), pages 728 - 735, XP037768966, [retrieved on 20220316], DOI: 10.1038/S41586-022-04494-7 *
LEGUTMATEUSZ ET AL., NATURE, vol. 603, no. 7902, 2022, pages 728 - 735
MACKENSEN ET AL., NAT. MED., vol. 29, 2023, pages 2844 - 2853
MILONE ET AL., MOL. THER., vol. 17, no. 8, 2009, pages 1453 - 1464
MIMITOU ET AL., NAT METHODS, vol. 16, no. 5, May 2019 (2019-05-01), pages 409 - 41
NISHIKAWA ET AL., HUM GENE THER, vol. 12, no. 8, 2001, pages 861 - 70
O'ROURKEDONALD MET, SCIENCE TRANSLATIONAL MEDICINE, vol. 9, no. 399, 2017, pages 0984
PARKLEE, EXP MOL MED, vol. 53, no. 3, March 2021 (2021-03-01), pages 318 - 327
QHTSUKA, BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
ROSSOLIM, MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98
SACK ET AL., CELL, vol. 173, no. 2, 5 April 2018 (2018-04-05), pages 499 - 514
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR PRESS
SANJANA, N. E., ANAL. BIOCHEM., vol. 532, 2017, pages 95 - 99
SCHMETTERER, K. G. ET AL., FRONT. IMMUNOL., vol. 10, 2019, pages 1790
SINGHNATHANMARCELA V MAUS, IMMUNITY, vol. 56, no. 10, 2023, pages 2296 - 2310
SOMMERMEYER, D. ET AL., LEUKEMIA, vol. 30, 2016, pages 492 - 500
STOECKIUS ET AL., GENOME BIOL, vol. 19, 2018, pages 224
STOECKIUS ET AL., NAT METHODS, vol. 14, no. 9, September 2017 (2017-09-01), pages 865 - 868
X. SU ET AL., MOL. PHARMACEUTICS, vol. 8, no. 3, 21 March 2011 (2011-03-21), pages 774 - 787

Similar Documents

Publication Publication Date Title
US12421557B2 (en) Methods for predicting outcomes and treating colorectal cancer using a cell atlas
US12006329B2 (en) Protein degraders and uses thereof
US20220025369A1 (en) Rna encoding a therapeutic protein
US12297428B2 (en) Compositions for identification of membrane targets for enhancement of T cell activity against cancer
US12281301B2 (en) Sequencing-based proteomics
US20210363525A1 (en) Sarna compositions and methods of use
US20230193205A1 (en) Gene modified fibroblasts for therapeutic applications
US20230203485A1 (en) Methods for modulating mhc-i expression and immunotherapy uses thereof
US20220401460A1 (en) Modulating resistance to bcl-2 inhibitors
US20240043934A1 (en) Pancreatic ductal adenocarcinoma signatures and uses thereof
US20210371932A1 (en) Methods and compositions for detecting and modulating microenvironment gene signatures from the csf of metastasis patients
US20240261333A1 (en) Novel targets for enhancing anti-tumor immunity
CN117730164A (en) Method for managing cell quality and method for producing cell
JP7162406B1 (en) Cell quality control method and cell manufacturing method
US12054756B2 (en) Engineered nucleases, compositions, and methods of use thereof
TW202307215A (en) Cell quality management method and cell production method
CN117677707A (en) Quality control method for specific cells and method for producing specific cells
TW202307203A (en) Method for managing quality of specific cells, and method for manufacturing specific cells
US20240307444A1 (en) Methods and compositions for improved immunotherapies
US10851370B1 (en) Homologous recombination directed genome editing in eukaryotes
JP2025510781A (en) Recombinant gene effectors and compositions and methods of use thereof
WO2025184170A1 (en) Methods and compositions for improving t cell function in an immunosuppressive environment
US20220106598A1 (en) Homologous recombination directed genome editing in eukaryotes
US20250388634A1 (en) Engineered gene effectors, compositions, and methods of use thereof
Curtis A genome-scale gain-of-function screening platform nominates genetic modulators for CAR T cell therapy enhancement

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25711394

Country of ref document: EP

Kind code of ref document: A1