Enhanced chimeric antigen receptor cells in hypoxic tumor microenvironment
Embodiments of the present disclosure relate to compositions and methods of enhancing lymphocytes' ability to treat cancer patients. Embodiments relate to a polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR), a nucleic acid encoding an Oxygen-Dependent Degradation domain (ODD), and a nucleic acid encoding one or multiple sequences of Hypoxia-Response Element (HRE).
1 . A method of maintaining a population of T cells expressing a chimeric antigen receptor (CAR) under hypoxic conditions, the method comprising: introducing a polynucleotide comprising a nucleic acid encoding an Oxygen-Dependent Degradation domain (ODD) comprising SEQ ID NO: 17 and a nucleic acid comprising one or more sequences of Hypoxia-Response Element (HRE) and a nucleic acid encoding the CAR into a population of T cells; and
exposing the population of T cells to a hypoxic condition, wherein maintenance of the population of T cells is higher than that of a population of T cells without the nucleic acid encoding the ODD and the nucleic acid comprising one or more sequences of HRE.
2 . The method of claim 1 , wherein the one or more sequences comprise nine repeated sequences of HRE.
3 . The method of claim 2 , wherein the one or more sequences comprise SEQ ID NO: 1.
4 . The method of claim 3 , wherein the CAR comprises SEQ ID NO: 6 and binds Fibroblast activation protein-α (FAP).
5 . The method of claim 3 , wherein the CAR comprises SEQ ID NO: 25 and binds guanylyl cyclase 2C (GUCY2C) or GCC.
6 . The method of claim 3 , wherein the CAR comprises SEQ ID NO: 21 and binds CLDN18.2.
7 . The method of claim 3 , wherein the CAR comprises SEQ ID NO: 23 and binds GPC3.
8 . The method of claim 3 , wherein the CAR binds to tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, CLDN18.2, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Rα2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, B7-H3, or EGFR.
9 . The method of claim 3 , wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
10 . The method of claim 9 , wherein the antigen binding domain binds to GCC, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, ML-IAP, TMPRSS2-ERG fusion protein, NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase (hTERT), RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1.
11 . The method of claim 9 , wherein the intracellular signaling domain comprises a signaling domain, or a primary signaling domain and a co-stimulatory signaling domain, and wherein the signaling domain or co-stimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.
12 . The method of claim 3 , wherein the population of T cells is engineered to express and secrete a therapeutic agent, and optionally wherein the therapeutic agent is a cytokine.
13 . The method of claim 12 , wherein the therapeutic agent comprises IL-6 or IFN-γ, or a combination thereof.
14 . The method of claim 12 , wherein the therapeutic agent comprises IL-15 or IL-12, or a combination thereof.
15 . The method of claim 12 , wherein the therapeutic agent is or comprises a recombinant or naturally occurring cytokine.
16 . The method of claim 3 , wherein the population of T cells is derived from a healthy donor or a subject having cancer.
17 . The method of claim 3 , wherein the population of T cells has a reduced expression of endogenous TRAC gene.
18 . The method of claim 3 , wherein the population of T cells further comprises an additional CAR binding a white blood cell antigen.
19 . The method of claim 18 , wherein the white blood cell antigen is CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13.