In Vivo Generation of CAR T Cells Selectively in Human CD4⁺ Lymphocytes

T cells modified with CD19-specific chimeric antigen receptors (CARs) result in significant clinical benefit for leukemia patients but constitute a challenge for manufacturing. We have recently demonstrated the in vivo generation of CD19-CAR T cells using the CD8-targeted lentiviral vector (CD8-LV). In this study, we investigated the in vivo generation of CD4⁺ CAR T cells using CD4-targeted LV (CD4-LV). Administration of CD4-LV into NSG mice transplanted with human peripheral blood mononuclear cells (PBMCs) led to 40%-60% of human CD4⁺ lymphocytes being CAR positive while CD8⁺ cells remained CAR negative. CAR⁺ T cells displayed a T helper 1 (Th1)/Th2 phenotype, which was accompanied by CD19⁺ B cell elimination. Intravenous administration of CD4-LV into NSG mice reconstituted with human CD34⁺ cells induced CAR expression and B cell elimination within 2-3 weeks post-injection. Preclinical analysis in a tumor mouse model revealed that mice administered CD4-LV exhibited faster and superior tumor cell killing compared to mice injected with CD8-LV alone or as a mixture with CD4-LV. Further analysis suggests that CD4⁺CAR⁺ cells may outperform CD8⁺CAR⁺ cells, especially at a high burden of target antigen, mainly since CD8 cells are more prone to exhaustion. This first description of in vivo-generated CD4⁺ CAR T cells supports their importance for cellular therapy.