A convenient viral transduction based method for advanced multi-engineering of primary human (CAR) T-cells

The past decades have illustrated the power of T-cell engineering in the development of new and successful cell therapies, such as chimeric antigen receptor (CAR) T-cells. Despite clinical success in hematological malignancies, it also becomes increasingly clear that additional T-cell engineering will be required to improve efficacy and safety and expand the application to solid tumors. Engineering is most often achieved by viral delivery of transgenes, however, viral vector capacity limitations make efficient and reproducible generation of multi transgene expressing T-cell therapeutics technically challenging. We here describe a convenient and efficient method for the delivery of up to three γ-retroviral CAR vectors in T-cells. We achieved this using virus vector mixtures that are simultaneously produced at high titers by double- or triple- transduced stable virus producer cells. We show that this method is superior in overall efficiency and reproducibility to conventional double or triple CAR transductions, in which separate viral batches are used. Due to its robustness, this method can facilitate the research and the development for advanced T-cell engineering towards more effective and safe therapies.