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

Anti-CD3/CD28 (aCD3/28) 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 aCD3/28-generated duoCAR-Ts, HCW9206-generated duoCAR-Ts were markedly enriched for TSCM (> 50%), displayed potent in vitro HIV-1 viral suppression, and antigen-induced TNFa and IFN? 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 aCD3/aCD28 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 aCD3/28 activation for producing functionally persistent CAR-Ts. Overall design: To evaluate the transcriptional differences between anti-HIV duoCAR-T cells generated by standard anti-CD3/CD28 stimulation (duoCAR-T-aCD3/28) and treatment with the novel human cytokine-based scaffold HCW9206 (duoCAR-T-HCW9206). The CAR-T transcriptomes were compared with or without stimulation with 293T cells expressing HIV-1 envelope protein (293T-gp120). CD8+ duoCAR-T-aCD3/28 cells and duoCAR-T-HCW9206 cells were cultured with 293T cells (unactivated) or cultured with 293T-gp120 cells (activated) for 16 hours. CD8+ duoCAR-T cells were purified by FACS, and total RNA was isolated. 2 biological replicates underwent treatment, cell sorting, RNA isolation, and sequencing independently.