Engineering Multiparametric Evaluation of Environmental Cues by Mammalian Cell-based Devices

Engineered cell-based therapies are a promising emerging strategy for overcoming existing barriers to treatment. Reaching the full potential of this powerful therapeutic strategy requires new tools for engineering mammalian cells to sense and respond to their physiological environment in programmable ways. In particular, the engineered cell should be able to 1) sense the cues in its environment and 2) evaluate multiple cues such that activation of its therapeutic function is conditional upon whether it senses a healthy or a diseased environment. This sensing and evaluation cascade should furthermore be performed in a manner orthogonal to the native signaling pathways of the cell, to avoid interference with or by these native pathways. Orthogonality also confers cell type independence, such that the technology could be ported into any cell type of interest with minimal modification. To meet these needs, we have previously described a platform technology to transduce an extracellular sensing event into a change in cell state. We have developed the first fully orthogonal cell surface biosensor platform, termed a modular extracellular sensor architecture (MESA), and we have described the engineering of a generic dimerization-dependent signal induction mechanism.;Here we present an expansion of that technology to activate alternative output modalities, to sense extracellular species via a novel single chain antibody-derived binding domain, and to perform multiparametric sensing and evaluation within mammalian cells. First we investigated whether the MESA could be configured to activate an alternative output, by reconstituting an enzyme in response to ligand-induced dimerization. Second, we investigated whether we could achieve sensing of exclusively extracellular ligands using the MESA platform. Leveraging a novel protein binding domain, the nanobody, we demonstrated MESA that transduce an extracellular ligand-binding event into an orthogonal intracellular signaling event. Moreover, we demonstrated that these protein-binding MESA are readily adaptable to recognizing a distinct cue, and that two MESA receptor pairs specific for distinct cues could be multiplexed into a logic gate for multiparametric evaluation of the extracellular environment. As a whole, this work fills an important gap in the mammalian synthetic biology toolbox and may enable novel therapeutic strategies using engineered cells.