Modular Receptor Engineering for Programming Cell-Based Therapies to Interface with Host Physiology

Engineered cell-based therapies represent an emerging frontier in medicine, and the promise of this approach has recently been demonstrated through curative eradication of B cell malignancy from a growing list of patients. However, extending this success to other types of cancer and to other applications in health and medicine will require new tools enabling bioengineers to custom program the manner in which engineered cells interface with host physiology. Until recently, we lacked the ability to construct customizable cell-based “devices” that detect and respond to exclusively extracellular cues, which include many species of biological relevance including cytokines, chemokines, cell-surface antigens, and pathogens. To meet this need, we developed a Modular Extracellular Sensing Architecture. This protein engineering platform comprises a self-contained receptor and signal transduction system, wherein ligand binding at the cell surface is transduced into a change in intracellular state (i.e., induction of gene expression) without requiring involvement of native intracellular signaling mediators, and therefore without being subject to native mechanisms regulating signal transduction. Here, we report our expansion upon this platform to enable the engineering of receptors that recognize essentially any external ligand and regulate essentially any native gene. We have developed general and rapidly employable engineering approaches for incorporating modular ligand binding domains (including both single-chain variable fragments, scFv, and single domain Nanobodies) to create new functional receptors. We have also engineered MESA receptors to generate “output” via Cas9-based gene regulation systems. Cas9-based transcriptional activators and repressors are easily targeted to regulate either native genes or engineered transgenes by co-expression with single-guide RNA (sgRNA) tailored to target a specific gene or set of genes. Finally, we have demonstrated that these approaches may be combined to functionally rewire mammalian cell input-output behavior. Ultimately, this capability provides a powerful tool for experimental systems biology, and in the context of medicine, enables the rapid evaluation of multiple potential strategies for functionally rewiring multicellular networks to achieve a therapeutic objective. We will present our application of this approach to modulating immune function, toward the goal of therapeutically modulating local immune states to overcome persistent barriers to immunotherapy of cancer.