Increased NFAT activity with dual CAR stimulation in CD19xCD22 CAR T-cells is associated with decreased exhaustion and improved survival.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is effective in treating B-cell malignancies, however relapse due to lack of CAR persistence and antigen-modulated escape remains common. Multiple strategies to simultaneously target CD19 and CD22 have been able to reduce antigen-modulated escape but not completely eliminate relapse. A bicistronic CAR construct consisting of a CD19 CAR incorporating the CD28 costimulatory domain paired with a CD22 CAR incorporating a 4-1BB costimulatory domain (CD19xCD22) demonstrated superior preclinical activity compared with other configurations and is currently under clinical investigation (NCT05098613, NCT05442515, NCT06559189). We hypothesized that simultaneous activation of CD28-containing and 4-1BB-containing CAR molecules not only allows for targeting of both antigens but creates a unique signal which enhances CAR T-cell function and efficacy. METHODS: We tested CD19xCD22 CAR T-cells generated from primary human T-cells against NALM6 with wild-type expression of CD19 and CD22 (CD19+/CD22+) or CRISPR/Cas9 knockout of one or both antigens (CD19+/CD22-, CD19-/CD22+, CD19-/CD22-) to interrogate the effect of dual-CAR stimulation on T-cell function, signaling, and in vivo efficacy in xenograft models. RESULTS: In vitro proliferation and cytokine production of CD19xCD22 CAR T-cells were primarily driven by activation of the CD19-28z CAR, however the CD22-BBz CAR drove equivalent cytotoxicity. Dual-CAR stimulation of CD19xCD22 CAR T-cells decreased leukemia relapse and improved survival in xenograft models. This increase in efficacy was associated with increased signaling through the phospholipase C-gamma 1 and nuclear factor of activated T-cells pathway after dual-CAR stimulation. Dual-CAR stimulation also led to decreased expression of markers associated with T-cell exhaustion in persistent CD19xCD22 CAR T-cells. CONCLUSIONS: Stimulation of both CAR molecules in a CD19xCD22 bicistronic CAR construct impacts downstream signaling events within the CAR T-cell and subsequently drives a more efficacious in vivo response with evidence of decreased exhaustion in persisting cells. These data suggest that bicistronic CAR platforms have the potential to not only target two antigens to prevent antigen-modulated escape but can be engineered to improve multiple facets of CAR T-cell biology, such as mitigating exhaustion, thereby overcoming multiple mechanisms known to drive relapse in current CAR T-cell therapies.