765 Contextual secretion of nanoscale interleukin (IL)-12 by CAR T cells for the treatment of cancer

BackgroundInterleukin(IL)-12 activates T cells and macrophages pivoting the switch that turns chronic into acute inflammation and results in cancer rejection. However, despite formidable antitumor effects in preclinical models, 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 purposefully induces minimally effective doses of IL-12 for autocrine activation of CAR-T.MethodsRB-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).ResultsRB-2-12 induced autocrine production of 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 (figure 1b).Abstract 765 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 byConditional autocrine release of nanoscale-dose p70/IL-12 by RB-2-12 resulting in enhanced IFN-γ production, cytotoxicity and proliferation in vitro after three days of exposure to FaDu cells (figure 1a). Constitutive high dose release of p70-IL-12 by HER2+ Fadu cells engineered to constitutively express IL-12 (FaDu/IL-12) and its effect on IFN-γ secretion, cytotoxicity and proliferation of conventional HER-2-specific CAR T-cells three days after exposure to FaDu or FaDu/IL-12 cancer cells (figure 1b). N.T. = non-transduced T cells; NOsg = cRB-2-12 CAR-T cells missing the sgRNAs for the two IL-12 subunits; IL12sg =