Co-transplantation of CAR-gene Engineered Precursor T Cells Leads To Suppressed T Cell Development and Increased NK Cell-based CAR-mediated Anti-leukemic Activity

CAR gene expression on precursor T cells (preTs) that have been generated in vitro from engineered hematopoietic stem cells may reduce the risk of leukemia relapse upon co-transplantation in an MHC-independent manner. Setting the expression of an anti-murine CD19 CAR under the control of an inducible promoter allowed time-dependent assessment of its impact on in vivo development. Adoptive transfer of engineered preTs along with T cell-depleted bone marrow resulted in potent anti-leukemia effects even in fully MHC-class I and II mismatched recipients. Anti-leukemia effects entirely relied on the in vivo development of CAR-expressing natural killer cells since NK cell depletion after transplantation resulted in a complete abrogation of anti-leukemic efficacy. Gene expression profiling revealed that enforced CAR expression on preTs led to an activated shift of transcriptional key factors to those being associated with NK cell development such as Nfil3, Id2, and Eomes. In contrast, CAR expression on preTs blocked thymic seeding completely and prevented further T cell development. The CAR-expressing NK cell progeny of transferred preTs persisted for up to 60 days post transplantation allowing for complete B-cell recovery thereafter. Taken together, we provide new functional insights for the use of CAR-engineered preTs allowing for potent anti-leukemia efficiency within a limited time window for on target/off tumor effects. Microarray data analysis of sorted CAR preTs and Ctrl preTs from in vitro culture (day 20 preTs previous to transplantation) and of their respective progenies harvested from the mouse on day 28 after transplantation were performed.