CAR-T cell therapy can be hampered by tumor escape mediated by antigen loss. One strategy to mitigate this limitation is to use CAR-T cells simultaneously targeting two antigens, although the best approach for creating multi-targeting CAR-T cells has not been established yet. We generated dual CAR-T cells by co-transduction of two lentiviral vectors expressing one CAR each. Considering that cellular resources are limited, we hypothesized that co-expression of two CARs in the same T-cell could compete to use the transcription and translational machinery.
Here, several combinations of CAR constructs were co-transduced in T-cells. We observed that, in most cases, one or both co-transduced CARs were expressed at lower levels on the T-cell membrane compared with individually transduced controls. Impaired CAR expression following co-transduction was exacerbated when increasing multiplicities of infection were used, indicating that co-transduced CARs compete for a finite pool of cellular resources. To elucidate which steps may be rate-limiting, first we assessed vector copy number. No differences in viral genomes were observed, suggesting integration is not a rate-limiting mechanism. By contrast, lower RNA levels were detected after co-transduction, suggesting the transcriptional machinery is sensitive to transgene expression burden. Finally, several codon optimized CAR versions and sequential transduction of the two lentiviral vectors were tested. However, both approaches failed to mitigate decreased CAR expression after co-transduction.
These preclinical results warrant further investigation to help refine the strategies currently being explored in the clinic using co-transduction to express two transgenes of interest.