The ability to engineer complex logic in human immune cells can greatly improve specificity and further unlock the potential of immunotherapy. Additionally, such a synthetic approach could provide insights into the governing principles of biocomputation in human immune cells. The chimeric antigen receptor (CAR), which is typically composed of a single chain variable fragment fused to signaling domains from T-cell receptor and co-stimulatory receptor, was developed to redirect T-cell specificity toward cancer cells, with very high efficacy against a few cancers4,5,6,7. Current conventional CAR designs, however, can only detect one antigen, and therefore have limited specificity. To improve specificity and control, several approaches have been explored to incorporate basic logic and control functions into CARs. For instance, CAR systems that are only active when two antigens are present on a cancer cell (AND gate) have been created8,9, whereby T cells are transduced with a CD3ζ CAR directed towards one antigen and a chimeric co-stimulatory receptor directed towards a second antigen. In conventional T cells, activation of both CD3ζ and a co-stimulatory signaling pathway (e.g., CD28 or 4-1BB) are needed to induce the full response to eradicate tumors. However, the activity of these combinatorial CAR systems cannot be tuned once the cells have been engineered. Furthermore, for the combinatorial CAR design to function effectively as an AND gate, the activity of each CAR needs to be carefully balanced8,9.
Since conventional CARs are functional in many immune cell types, we also tested whether the SUPRA CAR system can redirect antigen specificity in various T-cell subtypes, NK cell, and macrophage. These cell types were chosen because of their potential therapeutic applications. Here we will focus on evaluating the induciblity and logic operation of the SUPRA CAR system by measuring population-level cell killing for cytotoxic cells and cytokine production for other immune cells. These responses were chosen because of their clinical importance. All immune cells were lentivirally transduced to express zipCARs and co-cultured with or without α-Her2 zipFvs in the presence of the Her2-expressing Nalm6 target cells. Consistent with our previous report10, SUPRA CAR can efficiently induce target cell killing by CD8+ T cells (Fig. 1a). Furthermore, we differentiated naive CD4+ T cells to Th1 and Th2 cells to show that SUPRA CAR-expressing Th1 and Th2 cells can secrete IFN-γ and IL-4, respectively, when the corresponding zipFv was added (Figs. 1b, c, and Supplementary Fig. 1a). Additionally, regulatory T (Treg) cells are a unique subtype of CD4+ T cells that show various antigen-dependent immunosuppressive phenotypes, such as secretion of IL-10 and expression of CTLA-411, and have been strongly suggested to be clinically effective in treating autoimmune disorders12. A pan-T-cell activation marker, CD69, was upregulated in SUPRA CAR-containing primary human Treg cells when the corresponding zipFv was added (Fig. 1d, Supplementary Fig. 1b, and S1c).
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