Gene editing of naive and central memory T lymphocyte specificities for adoptive immune therapy of leukemia

Transfer of a T cell receptor (TCR) from a high-avidity tumor-specific T cell (CTLs) to polyclonal T cells may overcome the difficulty of expanding rare tumor-specific CTLs, under conditions that can preserve function and prevent exhaustion. Unfortunately, TCR-gene transfer is limited by: 1. Low and transient transgene expression; 2. Inappropriate pairing of the exogenous and endogenous TCR chains; 3. Poor survival and expansion of gene-modified effectors. We cloned genes encoding a high-avidity TCR specific for an HLA-A2-restricted pep-tide from the Wilms tumor antigen 1 (WT1), into a third generation lentiviral vector (LV) under the control of a bi-directional PGK or EF1a promoter. To increase TCR expression and facilitate appropriate TCR pairing, we used a codon-optimized TCR, modified with additional cysteines in the constant region of alpha and beta chains. T lymphocytes were efficiently transduced by both vectors following activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/ CD28) and culture with low doses of IL-7/IL-15. However, the PGK promoter was superior to EF1a in sustaining stochiomet-ric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 50 days after stimulation. We observed that early T cell differentiation phenotype (CD45RA-/+CD62L+, CD28+ CD27+, IL7Ra+, IL-2+ gIFN-/+), was preserved in TCR-modified lymphocytes. Sorted naive (CD45RA+/CD62L+) and central memory (CD45RA-/CD62L+) lymphocytes were efficiently transduced by TCR-LV and maintained the original phenotype. Upon antigenic stimulation, TCR-modified lymphocytes mediated WT1-specific gIFN production and cytotoxicity. To further improve the safety of the strategy, we attempted site-specific integration of transgenes by using of designed zinc finger nucleases (ZFN). Adenoviral transfer of a set of ZFN specific for CCR5 locus coupled with integrase defective lentiviral vectors carrying transgenes, enabled efficient site-specific integration in human lymphocytes resulting in stable transgene expression. Site-specific integration of optimized, leukemia-specific TCR into naive and central memory lymphocytes may represent an effective method for the generation of robust engineered tumor-specific CTLs.