Background Interleukin(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.

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 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).

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Abstract 765 Figure 1

Conditional 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 = complete product incorporating the full CRISPRa functions.

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.

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References

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