CAR T cell engineering impacts antigen-independent activation and co-inhibition

Viral vectors have successfully modified T cells to express chimeric antigen receptors (CARs), leading to clinical approvals. However, their high cost and regulatory challenges hinder rapid clinical translation. Here, we demonstrate that our lentivirally (LV) manufactured R110-CAR T cells, targeting a leukemia neoepitope, can also be engineered using the non-viral sleeping beauty (SB) transposition with minimal-sized DNA vectors. Flow cytometry and single-cell sequencing were used to compare the two production modes using healthy donor and chronic lymphocytic leukemia (CLL)-patient-derived T cells and a CD19-CAR T cell control. SB products shifted toward CD8⁺ subsets with activation/co-inhibition marker expression (CD69, LAG-3, and TIM-3) despite their naïve-like phenotype and lack of antigenic challenge. The CAR binding moiety modulated these patterns, with R110-CAR T cells showing more aberrant phenotypes. Moreover, SB engineering resulted in inflammatory signatures with RIG-I-like and TOLL-like nucleotide sensing potentially due to the transfection procedure. Patient-derived products showed fewer CAR-expressing cells, reduced proliferation clusters, and lower T cell diversity, particularly with SB manufacturing, indicating potential challenges with this method when engineering CLL T cells. Together, our data suggest that the engineering mode may substantially influence T cell properties and that these are further modulated by the CAR binding moiety and the T cell donor.