Chimeric antigen receptor (CAR)-modified T cells have revealed promising results in the treatment of cancer, but they still need to overcome various hurdles, including a complicated manufacturing process. Receptor-targeted lentiviral vectors (LVs) delivering genes selectively to T cell subtypes may facilitate and improve CAR T cell generation, but so far they have resulted in lower gene delivery rates than conventional LVs (vesicular stomatitis virus [VSV]-LV). To overcome this limitation, we studied the effect of the transduction enhancer Vectofusin-1 on gene delivery to human T cells with CD4-and CD8-targeted LVs, respectively, encoding a second-generation CD19-CAR in conjunction with a truncated version of the low-affinity nerve growth factor receptor (DLNGFR) as reporter. Vectofusin-1 significantly enhanced the gene delivery of CD4-and CD8-LVs without a loss in target cell selectivity and killing capability of the generated CAR T cells. Notably, delivery rates mediated by VSV-LV were substantially reduced by Vectofusin-1. Interestingly, a transient off-target signal in samples treated with Vectofusin-1 was observed early after transduction. However, this effect was not caused by uptake and expression of the transgene in off-target cells, but rather it resulted from cell-bound LV particles having DLNGFR incorporated into their surface. The data demonstrate that gene transfer rates in the range of those mediated by VSV-LVs can be achieved with receptor-targeted LVs.
Natural killer (NK) cells are a noteworthy lymphocyte subset in cancer adoptive cell therapy. NK cells initiate innate immune responses against infections and malignancies with natural cytotoxicity, which is independent of foreign antigen recognition. Based on these substantive features, genetically modifying NK cells is among the prime goals in immunotherapy but is currently difficult to achieve. Recently, we reported a fully human CAR19 construct (huCAR19) with remarkable function in gene-modified T-cells. Here, we show efficient and stable gene delivery of huCAR19 to primary human NK cells using lentiviral vectors with transduction efficiencies comparable to those achieved with NK cell lines. These huCAR19 NK cells display specific and potent cytotoxic activity against target cells. To improve homing of NK cells to the bone marrow, we augmented huCAR19 NK cells with the human CXCR4 gene, resulting in transgenically augmented CAR NK cells (TRACKs). Compared to conventional CAR NK cells, TRACKs exhibit enhanced migration capacity in response to recombinant SDF-1 or bone marrow stromal cells while retaining functional and cytolytic activity against target cells. Based on these promising findings, TRACKs may become a novel candidate for immunotherapeutic strategies in clinical applications.
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