Efficient Production of Chimeric Antigen Receptor (CAR) T Cells with Transgenes Exceeding 10 kb Using Lentiviral...

Engineering multiple receptors into chimeric antigen receptor (CAR) T cells has emerged as a powerful strategy to prevent antigen-negative relapse and reduce on-target/off-tumor toxicities. However, manufacturing multi-receptor CAR T cells remains challenging, as increasing lentiviral transgene sizes significantly reduces viral titers and T cell transduction rates. Current production workflows often rely on cell sorting to enrich transduced T cells from low-yield productions. Yet, cell sorting techniques do not increase the absolute number of CAR T cells and add further complexity to the already elaborate manufacturing process. Consequently, these limitations impede the clinical translation of multi-receptor CAR T cells and restrict the development of next-generation immunotherapies. Here, we present a detailed, step-by-step production workflow optimized for generating CAR T cells with large lentiviral transgenes. Using this workflow, we demonstrate size-dependent increases in transduction efficiency across a range of transgene sizes, with the most pronounced enhancement of up to 14.8-fold observed for a 10.1 kb lentiviral vector. Importantly, the workflow supports robust T cell expansion and eliminates the need for cell sorting. By overcoming current size limitations in lentiviral gene transfer, this workflow enables the efficient generation of multi-receptor CAR T cells, thereby facilitating the development of advanced immunotherapies.