Phosphoglycerate kinase 1 (pgk) promoters and methods of use for expressing chimeric antigen receptor

Abstract

The invention provides compositions and methods for treating diseases associated with expression of a tumor antigen as described herein. The invention also relates to nucleic acids comprising a truncated PGK promoter operably linked to a chimeric antigen receptor (CAR) specific to a tumor antigen as described herein, vectors encoding the same, and recombinant T cells comprising the CARs of the present invention. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises an antigen binding domain that binds to a tumor antigen as described herein.

1 - 48 . (canceled) 
     
     
         49 . A method for eliciting an inducible proliferation of an engineered immune cell, the method comprising
 transducing an immune cell with a lentiviral vector comprising:
 (a) a first nucleic acid sequence encoding a truncated PGK promoter comprising at least 100 nucleotides deletion at the 3′-end or the 5′-end of a wild-type (WT) PGK promoter set forth in SEQ ID NO: 1; and 
 (b) a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a 41BB, CD27 or CD28 costimulatory domain, and an intracellular signaling domain; 
   wherein the truncated PGK promoter modulates the transcription of the nucleic acid sequence encoding the CAR at a desired level, and modifies but retains the ability to direct expression of the CAR, thereby promoting the inducible proliferation of the engineered immune cell.   
     
     
         50 . The method of  claim 49 , wherein the CAR has an affinity (Kd) for its target antigen of less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.2 nM, or less than about 0.1 nM. 
     
     
         51 . The method of  claim 49 , wherein the CAR targets a tumor antigen that is also present on normal cells. 
     
     
         52 . The method of  claim 49 , wherein the engineered immune cell exhibits enhanced tumor infiltration when compared to an engineered immune cell transduced with a CAR operably linked to a wild-type (WT) PGK promoter or an elongation Growth Factor-1a (EF-1a) promoter. 
     
     
         53 . The method of  claim 49 , wherein the truncated PGK promoter comprises at least a 150, a 200, a 250, a 300, a 350, or a 400 nucleotide deletion in SEQ ID NO: 1. 
     
     
         54 . The method of  claim 49 , wherein the truncated PGK promoter consists essentially of:
 (a) a fragment of the WT PGK promoter nucleic acid sequence having a deletion at positions −521 to −422 of SEQ ID NO: 1, positions −521 to −324 of SEQ ID NO: 1, positions −521 to −221 of SEQ ID NO: 1, or positions −521 to −118 of SEQ ID NO: 1;   (b) nucleic acid sequences located at positions −1 to −422 of SEQ ID NO: 1, positions −1 to −324 of SEQ ID NO: 1, positions −1 to −221 of SEQ ID NO: 1, or positions −1 to −118 of SEQ ID NO: 1, or   (c) a nucleic acid sequence located at positions −521 to −422 of SEQ ID NO: 1.   
     
     
         55 . The method of  claim 49 , wherein the truncated PGK promoter consists essentially of a sequence at least 70% identical to a sequence selected from the group consisting of PGK100 (SEQ ID NO: 2); PGK200 (SEQ ID NO: 3); PGK300 (SEQ ID NO: 4); and PGK400 (SEQ ID NO:5). 
     
     
         56 . The method of  claim 49 , wherein the truncated PGK promoter consists essentially of PGK100 (SEQ ID NO: 2). 
     
     
         57 . The method of  claim 49 , wherein the antigen binding domain binds to a tumor antigen selected from the group consisting of: mesothelin, c-Met, 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, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, 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, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic 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, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fas-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), 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, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, c-met, and IGLL1. 
     
     
         58 . The method of  claim 49 , wherein:
 (a) the intracellular signaling domain is a CD3 zeta signaling domain; and/or   (b) the costimulatory domain comprises a CD28 costimulatory domain.   
     
     
         59 . The method of  claim 49 , wherein the immune cell is a human T cell, a human CD8 +  T cell, a human CD4 +  T cell, or a human NK cell. 
     
     
         60 . A cell engineered by the method of  claim 49 . 
     
     
         61 . A method of reducing one or more side effects associated with an administration of a CART cell, the method comprising administering to a subject the cell of  claim 60 . 
     
     
         62 . The method of  claim 61 , wherein the one or more side effects are associated with a higher CAR expression, the presence of a CAR target on normal cells, or a CAR with a high affinity for its target antigen. 
     
     
         63 . The method of  claim 62 , wherein the high affinity (Kd) is an affinity of less than about 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, or 0.1 nM. 
     
     
         64 . A method of treating a subject having a disease associated with expression of a tumor antigen, comprising administering to the subject the cell of  claim 60 . 
     
     
         65 . The method of  claim 64 , wherein the cell is autologous or allogeneic to the subject to be administered the cell. 
     
     
         66 . The method of  claim 64 , wherein the cell induces less side effects when compared to an engineered immune cell transduced with a CAR operably linked to a wild-type (WT) PGK promoter or an elongation Growth Factor-1α (EF-1α) promoter. 
     
     
         67 . The method of  claim 64 , wherein the subject has a cancer selected from the group consisting of B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CIVIL), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and any combination thereof. 
     
     
         68 . A lentiviral vector comprising:
 (a) a first nucleic acid sequence encoding a truncated PGK promoter comprising at least 100 nucleotides deletion in a wild-type (WT) PGK promoter set forth in SEQ ID NO: 1; and   (b) a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a 41BB, CD27 or CD28 costimulatory domain, and an intracellular signaling domain;   wherein when expressed in an immune cell, the truncated PGK promoter modulates the transcription of the nucleic acid sequence encoding the CAR at a desired level, and modifies but retains the ability to direct expression of the CAR.   
     
     
         69 . A lentiviral vector comprising:
 (a) a first nucleic acid sequence encoding a truncated PGK promoter consists essentially of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence selected from the group consisting of PGK100 (SEQ ID NO: 2); PGK200 (SEQ ID NO: 3); PGK300 (SEQ ID NO: 4); and PGK400 (SEQ ID NO:5); and   (b) a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a 41BB, CD27 or CD28 costimulatory domain, and an intracellular signaling domain;   wherein when expressed in an immune cell, the truncated PGK promoter modulates the transcription of the nucleic acid sequence encoding the CAR at a desired level, and modifies but retains the ability to direct expression of the CAR.