Nanoscale, Antigen-Dependent, Reversible Il-12 Secretion By Car T Cells For Cancer Treatment.

Abstract BACKGROUND - Interleukin(IL)-12 activates T cells pivots the switch that turns lingering inflammation into acute inflammation and cancer rejection. However, its clinical utilization is limited by severe systemic toxicity. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-2-12) that induces minimally effective doses of IL-12 for autocrine activation of CAR-T cells. METHODS - RB-2-12 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 a lentiviral constructs encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the promoter region for IL-12A orL-12B. A second constructs encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR brings CAR-TEV in proximity to LdCV releasing dCas9 for nuclear localization to the regulatory regions and conditionally and reversibly induce nanoscale expression of the p70 heterodimer. RB-2-12 was compared in vitro to control (cRB-2-12, lacking the IL-12 sgRNAs). Results - RB-2-12 induced low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells resulting in significantly enhanced production of interferon (IFN)-γ, cytotoxic activity and proliferation (Figure 1a). These effects were comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line expressing high doses of IL-12. In vivo administration of RB-2-12 significantly enhanced survival of mice carrying FaDu xenografts compared to mice treated with the respective conventional CAR-t cells or RB-2-12 lacking the guide RNAs targeting the IL-12 gene subunits CONCLUSIONS - We have previously shown that tandem suppression of PD-1 expression upon HER-2 CAR activation using CRISPR interference enhances anti-cancer properties of CAR-T cells in vivo against HER2-FaDu xenografts by promoting their persistence and long-term tumor colonization (companion abstract submitted to SITC annual meeting). We hypothesize that addition of a Th1 polarizing component such as IL-12 will exponentially increase the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. At the same time, the autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Such cumulative synthetic biology approaches are currently investigated in vitro and in vivo model systems. Current work is testing the effectiveness of RB-2-12 in vivo against FaDu xenografts. Citation Format: Zhifen Yang, Maggie Bobbin, Hana Choi, Ofir Stefanson, Khristina Magallanes, Jin Yang, Bing Wang, Alessandra Cesano, Lei Stanley Qi, Francesco M. Marincola. Nanoscale, antigen-dependent, reversible IL-12 secretion by CAR T cells for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB026.