ARTICLES https://doi.org/10.1038/s41589-018-0046-z 1 Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 2 University of Basel, Faculty of Science, Basel, Switzerland. *e-mail: fussenegger@bsse.ethz.ch M ammalian cells programmed to respond to extracellular inputs in a predictable manner have become increasingly important for a wide range of applications, such as cancer immunotherapy, tissue patterning and smart cell implants. Indeed, the field of programmable receptor engineering is rapidly evolv- ing 1,2 , but robust sensing of soluble molecules still mostly relies on natural receptors that can be rewired to drive expression of trans- genes that have a desired biological function. For example, natural ligand–receptor interactions have been used to engineer designer cells that sense various biomarkers and secrete therapeutic pep- tides in response. This approach has been used to develop thera- peutic cell implants consisting of encapsulated designer cells for the detection and treatment of psoriasis, Graves’ disease and meta- bolic syndrome 3–5 . Nevertheless, engineering robust input–output relationships in mammalian cells is a laborious iterative process, and many molecules that would be valuable targets for diagnostic or therapeutic purposes do not bind to any naturally occurring receptor. Thus, large groups of potential molecular inputs cannot be targeted by this approach. Notably, this includes the majority of synthetic small-molecule compounds, intracellular proteins and extracellular proteins without signaling function. To overcome the limitations of natural receptors, scFvs (single- chain variable fragments) of antibodies have been linked to extra- cellular domains of different receptors to generate customizable epitope sensors. Nonlimiting examples include CARs (chimeric antigen receptors) consisting of scFvs linked to T-cell receptors that can be used to generate T-cells with enhanced tumor targeting 6 . A synthetic notch receptor (SynNotch) 7 and cytokine-receptor- based T-cell-receptor-like signaling 8 have been engineered, enabling cell contact-based, antigen-dependent signaling. The MESA (mod- ular extracellular sensor architecture) system is based on bring- ing intracellular membrane-anchored TEV (tobacco etch virus) protease into proximity to a membrane-anchored transcription factor in response to extracellular ligand binding. This is followed by cleavage and nuclear localization of the transcription factor and has been used to sense human VEGF (vascular endothelial growth factor) 9 . In this study, we present a novel strategy to design a highly gen- eralizable modular platform for sensing and responding to extra- cellular molecules by using EpoR (erythropoietin receptor) dimers combined with different affinity domains, as well as three differ- ent intracellular domains for rerouting signaling to activate distinct endogenous signaling pathways. We have successfully rewired each of these pathways to transgene expression in human cells. This setup provides a robust platform, which we designate as Generalized Extracellular Molecule Sensor (GEMS), for constructing sensors to detect a wide range of targets. We confirmed the validity and gen- eralizability of the GEMS platform by constructing sensors for non- signaling molecules with a wide range of molecular weights, namely an azo dye (RR120), nicotine, a peptide tag fused to mCherry (SunTag) and PSA (prostate-specific antigen), which is a clinically important cancer biomarker. These GEMS devices are the first examples of sensing soluble molecules with scFv-coupled recep- tors that utilize natural signaling cascades rewired for transgene expression. Additionally, we show that orthogonal GEMS signal- ing pathways can be multiplexed for two-input two-output systems and that GEMS can be used to modulate intracellular signaling cas- cades in immune cells. The easy adaptability of the GEMS platform to new targets should make it a useful tool for many applications in synthetic biology and for developing novel precision-guided cell-based therapeutics. Results Detailed description of the GEMS system. The GEMS system functions by the well-investigated mechanism of dimerization of extracellular receptor domains, which causes activation of intracel- lular signaling domains (Supplementary Fig. 1a). Cytokine recep- tors have a modular structure that tolerates the combination of intracellular and extracellular domains of different receptors to pro- duce functional chimeras 10,11 . Inactive EpoR dimers are locked by transmembrane helix interactions in a conformation that prevents downstream signaling 12 . Ligand binding to the receptors is thought to rotate each receptor subunit around its own axis and is likely accompanied by an increase in the distance between intracellular Generalized extracellular molecule sensor platform for programming cellular behavior Leo Scheller  1 , Tobias Strittmatter  1 , David Fuchs  1 , Daniel Bojar  1 and Martin Fussenegger  1,2 * Strategies for expanding the sensor space of designer receptors are urgently needed to tailor cell-based therapies to respond to any type of medically relevant molecules. Here, we describe a universal approach to designing receptor scaffolds that enables antibody-specific molecular input to activate JAK/STAT, MAPK, PLCG or PI3K/Akt signaling rewired to transgene expression driven by synthetic promoters. To demonstrate its scope, we equipped the GEMS (generalized extracellular molecule sensor) platform with antibody fragments targeting a synthetic azo dye, nicotine, a peptide tag and the PSA (prostate-specific anti- gen) biomarker, thereby covering inputs ranging from small molecules to proteins. These four GEMS devices provided robust signaling and transgene expression with high signal-to-noise ratios in response to their specific ligands. The sensitivity of the nicotine- and PSA-specific GEMS devices matched the clinically relevant concentration ranges, and PSA-specific GEMS were able to detect pathological PSA levels in the serum of patients diagnosed with prostate cancer. 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