Generation of persistent HIV CAR-TSCM cells by treatment with a novel common-gamma-chain cytokine-based fusion molecule

Anti-CD3/CD28 (αCD3/28) activation efficiently enables T cells to be transformed by transduction with CAR-expressing lentiviral vectors (LV) into chimeric antigen receptor (CAR)-T cells. However, the long-term effectiveness of this approach is compromised by the more differentiated phenotype of these CAR-Ts, associated with reduced in vivo functional persistence. Treatment with IL-7, IL-15, and/or IL-21 expands CAR-Ts with a T-stem-cell (TSCM) phenotype. We investigated whether we could use an alternative strategy to generate CAR-Ts, by using a scaffold-linked IL-7, IL-15, and IL-21 (HCW9206) to render T cells permissive for LV transduction and thereby promote the generation of HIV-specific duoCAR-T cells (duoCAR-T) enriched in a TSCM phenotype to enhance in vivo persistence. Compared to αCD3/28-generated duoCAR-Ts, HCW9206-generated duoCAR-Ts were markedly enriched for TSCM (> 50%), displayed potent in vitro HIV-1 viral suppression, and antigen-induced TNFα and IFNγ production. RNA sequencing analysis of the transcriptome of duoCAR-T cells generated by IL-7, IL-15, and IL-21 treatment delivered by HCW9206, compared to αCD3/αCD28 activation, identified multiple uniquely expressed genes. These results suggested that duoCAR-T-HCW9206 cells are enriched in both a TSCM phenotype, as indicated by pro-survival and TSCM-like gene signatures, as well as more differentiated effector-like phenotypes, likely contributing to the potent HIV suppression observed both in vitro and in vivo, despite primarily displaying a TSCM phenotype. Our results indicate that scaffold-linked IL-7, IL-15, and IL-21 fusion-treatment represents an alternative method for CAR-T cell generation, more effective than standard αCD3/28 activation for producing functionally persistent CAR-Ts.