Despite recent advances in mammalian synthetic biology, there remains a lack of modular synthetic receptors that can robustly respond to soluble ligands and, in turn, activate bespoke cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but so far only receptors against cell-surface targets have approached clinical translation. To address this gap, here we adapt a receptor architecture called the synthetic intramembrane proteolysis receptor (SNIPR) for activation by soluble ligands. Our SNIPR platform can be activated by both natural and synthetic soluble factors, with notably low baseline activity and high fold activation, through an endocytic, pH-dependent cleavage mechanism. We demonstrate the therapeutic capabilities of the receptor platform by localizing the activity of chimeric antigen receptor (CAR) T cells to solid tumours in which soluble disease-associated factors are expressed, bypassing the major hurdle of on-target off-tumour toxicity in bystander organs. We further apply the SNIPR platform to engineer fully synthetic signalling networks between cells orthogonal to natural signalling pathways, expanding the scope of synthetic biology. Our design framework enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

Clinical resistance to rat sarcoma virus (Ras)-G12C inhibitors is a challenge. A subpopulation of cancer cells has been shown to undergo genomic and transcriptional alterations to facilitate drug resistance but the immediate adaptive effects on Ras signaling in response to these drugs at the single-cell level is not well understood. Here, we used Ras biosensors to profile the activity and signaling environment of endogenous Ras at the single-cell level. We found that a subpopulation of KRas-G12C cells treated with Ras-G12C-guanosine-diphosphate inhibitors underwent adaptive signaling and metabolic changes driven by wild-type Ras at the Golgi and mutant KRas at the mitochondria, respectively. Our Ras biosensors identified major vault protein as a mediator of Ras activation through its scaffolding of Ras signaling pathway components and metabolite channels. Overall, methods including ours that facilitate direct analysis on the single-cell level can report the adaptations that subpopulations of cells adopt in response to cancer therapies, thus providing insight into drug resistance.

The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.