OFF-switch CAR T cell for safety-enhanced cancer immunotherapy

CARs are hybrid molecules comprising a tumor antigen-targeting moiety, typically a scFv, followed by a linker, transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. First generation CARs include the ED of CD3ζ only, required for ‘signal 19 of T cell activation, while 2 nd and 3 rd generation CARs also have one or more co-stimulatory EDs, respectively, such as CD28 and 41BB, to provide ‘signal 29. CAR T cells have shown robust clinical responses against advanced haematologial malignancies, leading to two recent FDA approvals for CD19-targeting CARs. Solid tumors, however, remain an important challenge to CAR therapy, in part due to the fact there are limited cell surface-expressed antigens (protein, carbohydrate, etc.) that are tumor-restricted, thus running the risk of severe on-target/off-tumor toxicity. Here we describe our progress in the development of a split OFF-switch CAR for which a small molecule is used to stop signaling. We designed a split CAR in which CD3ζ and the co-stimulatory ED are found on separate, co-expressed components. Briefly, Component A includes the scFv, a linker, TM domain, Protein A, and CD28, while the second one, Component B comprises a short extracellular region having no tumor-binding capacity, a TM domain, Protein B, a high affinity binding partner for Protein A, and CD3ζ. Proteins A and B were computationally designed from human proteins that normally do not interact, and upon the design of a novel protein-protein interaction (PPI) they form a heterodimer with an experimentally determined K D of 400 pM. Notably, this novel PPI was rationally designed to be rapidly disrupted by a clinically approved small molecule (K D of 10 pM for Protein B). Lentiviral constructs were built encoding the two components separately, or together on the same vector, and used to engineer both a Jurkat NFAT promoter-mCherry reporter line, and primary human T cells following activation with anti-CD3/anti-CD28 beads. Flow cytometry was used to assess CAR cell-surface expression, and AMNIS imaging to visualize co-localization. Both Jurkat and T cells could co-express Components A and B, but when transduced on their own, neither A nor B could be detected on the cell-surface. However, we observed instability of Component B over time. Finally, we demonstrated co-localization of the chains in Jurkats, as well as functionality of the CAR, as measured by mCherry and IL2 production, in the presence of target cells. Ongoing work includes the titration of the OFF-switch small molecule with target cells in vitro, and in vivo. We have also re-designed Component B to include a larger extracellular domain in an attempt to stabilize its expression. In conclusion, we used computational design to engineer a split-architecture CAR that can specifically activate engineered cells but further optimization is required to enhance its stability. We believe that such OFF-switch CARs hold important promise for increasing patient safety. Citation Format: Elise F. Gray-Gaillard, Greta Giordano Attianese, Pablo Gainza-Cirauqui, Sabrina Vollers, Sailan Shui, Bruno Correia, Melita Irving, George Coukos. OFF-switch CAR T cell for safety-enhanced cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2570.