T cell modifying compounds and uses thereof

Abstract

Methods and compositions for modifying T-cells in which PD1 and/or CTLA-4 is repressed and/or inactivated using fusion proteins such as artificial transcription factors and nucleases.

What is claimed is: 
     
         1 . A genetically modified T-cell that expresses a chimeric antigen receptor (CAR), wherein an exogenous sequence encoding a CAR is integrated into the genome of the T-cell, and further wherein expression of at least one endogenous immunological checkpoint gene is repressed by genetically modifying the immunological checkpoint gene in the T-cell, wherein the immunological checkpoint gene is a programmed death receptor PDCD1 gene or a CTLA-4 gene. 
     
     
         2 . The T-cell of  claim 1  wherein the immunological checkpoint gene is a CTLA-4 gene. 
     
     
         3 . The T-cell of  claim 1 , wherein the immunological checkpoint gene is PDCD1. 
     
     
         4 . The T-cell of  claim 1 , wherein the T-cell is selected from the group consisting of a CD4+ cell, a CD8+ cell and a tumor infiltrating cell (TIL). 
     
     
         5 . The T-cell of  claim 1 , wherein the exogenous sequence encoding the CAR is integrated into the T-cell genome at a safe harbor locus. 
     
     
         6 . The T-cell of  claim 1 , wherein the exogenous sequence encoding the CAR is randomly integrated into the T-cell genome. 
     
     
         7 . The T-cell of  claim 1 , wherein the CAR comprises a signaling domain of a T-cell receptor (TCR). 
     
     
         8 . The T-cell of  claim 7 , wherein the CAR comprises a scFv specificity domain. 
     
     
         9 . The T-cell of  claim 1 , further comprising at least one additional transgene. 
     
     
         10 . The T-cell of  claim 9 , wherein the at least one additional transgene encodes a tumor-associated antigen (TAA)-specific T-cell receptor (TCR). 
     
     
         11 . The T-cell of  claim 1 , wherein the T-cell is stimulated. 
     
     
         12 . The T-cell of  claim 11 , wherein the T-cell is stimulated with anti-CD28/CD3 beads. 
     
     
         13 . A method of making the T-cell of  claim 5 , the method comprising:
 cleaving the safe harbor gene and the at least one immunological checkpoint gene in the T-cell using one or more nucleases such that the exogenous sequence encoding the CAR is integrated into the safe harbor gene and the at least one immunological checkpoint gene is inactivated.   
     
     
         14 . The method of  claim 13 , where the safe harbor gene is selected from the group consisting of AAVS1, CCR5, HPRT and Rosa. 
     
     
         15 . The method of  claim 13 , wherein the exogenous sequence encoding the CAR is carried by a plasmid vector or a viral vector. 
     
     
         16 . The method of  claim 13 , wherein the nucleases are introduced into the cell as mRNA. 
     
     
         17 . The method of  claim 13 , wherein the T-cell is selected from the group consisting of a CD4+ cell, a CD8+ cell and a tumor infiltrating cell (TIL). 
     
     
         18 . The method of  claim 13 , wherein the CAR comprises a signaling domain of a T-cell receptor (TCR). 
     
     
         19 . The method of  claim 13 , wherein the CAR comprises a scFv specificity domain. 
     
     
         20 . The method of  claim 13 , further comprising stimulating the T-cell. 
     
     
         21 . The method of  claim 13 , wherein the T-cell is stimulated with anti-CD28/CD3 beads. 
     
     
         22 . The method of  claim 13 , further comprising integrating at least additional transgene into the T-cell genome. 
     
     
         23 . The method of  claim 22 , wherein the at least one additional transgene encodes a TAA-specific T-cell receptor (TCR). 
     
     
         24 . A method of making the T-cell of  claim 6 , the method comprising:
 cleaving the at least one immunological checkpoint gene using one or more nucleases such that the at least one immunological checkpoint gene is inactivated; and   randomly integrating the exogenous sequence encoding the CAR into the genome.