RT Journal Article SR Electronic T1 Coopting T cell proximal signaling molecules enables Boolean logic-gated CAR T cell control JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.06.17.496457 DO 10.1101/2022.06.17.496457 A1 Aidan M. Tousley A1 Maria Caterina Rotiroti A1 Louai Labanieh A1 Lea Wenting Rysavy A1 Skyler P. Rietberg A1 Eva L. de la Serna A1 Guillermo Nicolas Dalton A1 Dorota Klysz A1 Evan W. Weber A1 Won-Ju Kim A1 Peng Xu A1 Elena Sotillo A1 Alexander R. Dunn A1 Crystal L. Mackall A1 Robbie G. Majzner YR 2022 UL http://biorxiv.org/content/early/2022/06/17/2022.06.17.496457.abstract AB 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 cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3–7; however, a truly safe and effective logic-gated CAR has remained elusive8. 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 autoimmunity9 and fibrosis10. 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 StatementA.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.