Background Combining 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 eukaryotes^1–3 (RB-340-1) to promote CAR T resilience to checkpoint suppression extending in vivo persistence and effectiveness.

Methods RB-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).

Results RB-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.

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Abstract 142 Figure 1

Rb-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 with PDL1 blockade (Atezolizumab) is shown as dashed line. Colonization of tumors by human CD45+ cells is shown at the bottom of figure 1b & 1c

Conclusions Intrinsic conditional regulation of checkpoint expression in CAR T cells provides a simplified approach to combination therapies that limits systemic toxicities and reduces costs. Since the expression of multiple genes can be simultaneously controlled by CRISPRi, broader applications can be envisioned in the future.

Ethics Approval Not Applicable

Consent Not applicable

References

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2. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442–51.

3. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152(5):1173–83.