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.
Chimeric antigen receptor (CAR) expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype composition. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe here a novel receptor-targeted lentiviral vector (LV), named 62L-LV, that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4 and 50% of CD8 primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered binding of 62L-LV particles to T cells nor impacted their transduction. Incubation of two days activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in an NSG mouse model. The data prove that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selection of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.
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