Coopting T cell proximal signaling molecules enables Boolean logic-gated CAR T cell control

While CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells[1][1],[2][2]. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity[3][3]–[7][4]; however, a truly safe and effective logic-gated CAR has remained elusive[8][5]. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3ζ domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumors in vivo while bypassing upstream signaling proteins such as CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineer Logic-gated Intracellular NetworK (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity[9][6] and fibrosis[10][7]. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering. ### Competing Interest Statement A.M.T., R.G.M., M.C.R., L.L., and C.L.M. are inventors on a pending patent application for the novel CARs described in this manuscript. R.G.M., C.L.M., and L.L. are co-founders of and hold equity in Syncopation Life Sciences. R.G.M. and C.L.M. are cofounders of and hold equity in Link Cell Therapies. C.L.M. is a cofounder of and holds equity in Lyell Immunopharma. R.G.M, L.L., and E.W.W. are consultants for and hold equity in Lyell Immunopharma. A.M.T. is a consultant for Syncopation Life Sciences. S.R. is a former employee of and holds equity in Lyell Immunopharma. R.G.M. is a consultant for NKarta, Arovella Pharmaceuticals, Illumina Radiopharmaceuticals, GammaDelta Therapeutics, Aptorum Group, and Zai Labs. A.M.T. is a consultant for Syncopation Life Sciences. E.W.W. is a consultant for and holds equity in VISTAN Health. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-7 [5]: #ref-8 [6]: #ref-9 [7]: #ref-10