Background Interleukin(IL)-12 activates T cells pivoting the switch that turns lingering inflammation into acute inflammation and cancer rejection. However, its clinical utilization is limited by severe systemic toxicity. IL-12 is a potent inducer of PD-1 expression in T cells. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-312) that delivers nanoscale doses of IL-12 for autocrine activation of CAR-T cells. RB-312 was also tested in combination with PD-L1 blocking antibody (atezolizumab).
Methods RB-312 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes two lentiviral constructs with one encoding HER2-specific chimeric antigen receptor and two sgRNAs targeting IL-12A or IL-12B and the other encoding linker for activation of T cells, complexed to dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and hence conditionally and reversibly induces the secretion of IL-12 p70 heterodimer.
Results RB-312 induced low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells engineered to overexpress PD-L1 and this resulted in significantly enhanced production of IFN-γ, cytotoxicity and CAR-T proliferation (figure 1A). These effects were comparable to co-culturing conventional HER2 CAR with FaDu cells modified to express high doses of IL-12 (figure 1B). In vivo administration of RB-312 significantly enhanced survival of mice carrying FaDu xenografts compared to mice treated with the respective conventional HER2 CAR or cRB-312 (control lacking the IL-12 sgRNAs, figure 2A). RB-312 induced a strong upregulation of PD-1 in CAR-T cells in vivo (figure 2B). The critical role of the PD-1/PD-L1 interaction was demonstrated in vitro by comparing RB-312 proliferation when exposed to FaDu overexpressing PD-L1 or PD-L1 knock out cells (figure 3A). Indeed, combined treatment of RB-312 and atezolizumab resulted in significant reduction in tumor growth (figure 3B and C) and significantly enhanced survival (figure 3D).
Conclusions We concluded that addition of a Th1 polarizing component such as IL-12 exponentially increases the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. The autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Here, we tested the combination of PD-1/PD-L1 blockade with IL-12-induced CAR-T cell activation demonstrated dramatic synergistic effects. We are currently evaluating the intrinsic combination of IL-12 delivery and PD-L1 resistance for the next generation of RB-312 product eliminating the need for systemic checkpoint blockade.
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