142 Contextual reprogramming of CAR T cells for the treatment of HER2-expressing cancers

BackgroundCombining checkpoint inhibition (CPI) to adoptive cell therapy (ACT) is a promising strategy to prevent chimeric antigen receptor (CAR)-engineered T cell exhaustion and improve outcomes. However, cumulative toxicities and costs limit this approach. Here, we apply a conditional, antigen-dependent non-editing CRISPR interference-(CRISPRi) modulation circuit that we originally described in yeast and eukaryotes1–3 (RB-340-1) to promote CAR T resilience to checkpoint suppression extending in vivo persistence and effectiveness.MethodsRB-340-1 is an CAR T cell product engineered via synthetic biology approaches to express a combination of molecules to prevent CAR T cell exhaustion and improve solid tumor treatment outcomes. The components include two constructs. The first construct encodes an anti-HER2 (4D5) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a programmed cell death protein 1 (PD1) promoter region-targeting single guide RNA (PD1sg). The second construct encodes a protein, linker for activation of T cells (LAT), complexed to nuclease-deactivated/dead Cas9 (dCas9)-Kruppel-associated box (Krab) via a TEV-cleavable linker. Activation of CAR brings CAR-TEV in close proximity to the LAT-dCas9-Krab complex releasing the enzyme for nuclear localization to the PD1 regulatory region to conditionally and reversibly suppress its expression. RB-340-1 was compared in vitro and in vivo against conventional and control (cRB-340-1, lacking PD1sg) HER2 CAR T cells in combination with CPI with Atezolizumab (5 mg/Kg administered intravenously twice a week).ResultsRB-340-1 consistently induced higher production of homeostatic cytokines such as IL-2 resulting in significantly enhanced proliferation in vitro (figure 1a). Our in vivo data showed significantly enhanced suppression of growth of HER2+ FADU oropharyngeal cancer xenografts upon intra-tumoral (figure 1b) and systemic administration (figure 1c) and prolonged persistence of CAR T cells in vivo.Abstract 142 Figure 1Rb-340-1 performance in vitro and in vivoRB-340-1 (orange) decreased PD-1 expression resulting in enhanced cytokine production and proliferation in vitro (figure 1a) and superior tumor suppression in vivo after intra-tumoral (figure 1b) or intravenous (figure 1c) administration compared to conventional CAR T cells (blue) or cRB-340-1 (green). Conventional CAR T cells or cRB-340-1 CAR T combination treatment wi