Multiplexing Engineered Receptors for Multiparametric Evaluation of Environmental Ligands.

Engineered cell-based therapies comprise a promising, emerging biomedical technology. Broad utilization of this strategy will require new approaches for implementing sophisticated functional programs, such as sensing and responding to the environment in a defined fashion. Toward this goal, we investigated whether our self-contained receptor and signal transduction system (MESA) could be multiplexed to evaluate extracellular cues, with a focus on elucidating principles governing the integration of such engineered components. We first developed a set of hybrid promoters that exhibited AND gate activation by two transcription factors. We then evaluated these promoters when paired with two MESA receptors and various ligand combinations. Unexpectedly, although the multiplexed system exhibited distinct responses to ligands applied individually and in combination, the same synergy was not observed as when promoters were characterized with soluble transcription factors. Therefore, we developed a mechanistic computational model leveraging these observations, to both improve our understanding of how the receptors and promoters interface and to guide the design and implementation of future systems. Notably, the model explicitly accounts for the impact of intercellular variation on system characterization and performance. Model analysis identified key factors that affect the current receptors and promoters, and enabled an in silico exploration of potential modifications that inform the design of improved logic gates and their robustness to intercellular variation. Ultimately, this quantitative design-driven approach may guide the use and multiplexing of synthetic receptors for diverse custom biological functions beyond the case study considered here.

Rewiring human cellular input-output using modular extracellular sensors.

Engineered cell-based therapies comprise a promising emerging strategy for treating diverse diseases. Realizing this promise requires new tools for engineering cells to sense and respond to soluble extracellular factors, which provide information about both physiological state and the local environment. Here, we report such a biosensor engineering strategy, leveraging a self-contained receptor-signal transduction system termed modular extracellular sensor architecture (MESA). We developed MESA receptors that enable cells to sense vascular endothelial growth factor (VEGF) and, in response, secrete interleukin 2 (IL-2). By implementing these receptors in human T cells, we created a customized function not observed in nature-an immune cell that responds to a normally immunosuppressive cue (VEGF) by producing an immunostimulatory factor (IL-2). Because this platform utilizes modular, engineerable domains for ligand binding (antibodies) and output (programmable transcription factors based upon Cas9), this approach may be readily extended to novel inputs and outputs. This generalizable approach for rewiring cellular functions could enable both translational applications and fundamental biological research.

Engineering cell-based therapies to interface robustly with host physiology.

Engineered cell-based therapies comprise a rapidly growing clinical technology for treating disease by leveraging the natural capabilities of cells, including migration, information transduction, and biosynthesis and secretion. There now exists a substantial portfolio of intracellular and extracellular sensors that enable bioengineers to program cells to execute defined responses to specific changes in state or environmental cues. As our capability to construct more sophisticated cellular programs increases, assessing and improving the degree to which cell-based therapies perform as desired in vivo will become an increasingly important consideration and opportunity for technological advancement. In this review, we seek to describe both current capabilities and potential needs for building cell-based therapies that interface with host physiology in a manner that is robust - a phrase we use in this context to describe the achievement of therapeutic efficacy across a range of patients and implementations. We first review the portfolio of sensors and outputs currently available for use in cell-based therapies by highlighting key advancements and current gaps. Then, we propose a conceptual framework for evaluating and pursuing robust clinical performance of engineered cell-based therapies.

Contributions of unique intracellular domains to switchlike biosensing by Toll-like receptor 4.

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-ß (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.

Modular extracellular sensor architecture for engineering mammalian cell-based devices.

Engineering mammalian cell-based devices that monitor and therapeutically modulate human physiology is a promising and emerging frontier in clinical synthetic biology. However, realizing this vision will require new technologies enabling engineered circuitry to sense and respond to physiologically relevant cues. No existing technology enables an engineered cell to sense exclusively extracellular ligands, including proteins and pathogens, without relying upon native cellular receptors or signal transduction pathways that may be subject to crosstalk with native cellular components. To address this need, we here report a technology we term a Modular Extracellular Sensor Architecture (MESA). This self-contained receptor and signal transduction platform is maximally orthogonal to native cellular processes and comprises independent, tunable protein modules that enable performance optimization and straightforward engineering of novel MESA that recognize novel ligands. We demonstrate ligand-inducible activation of MESA signaling, optimization of receptor performance using design-based approaches, and generation of MESA biosensors that produce outputs in the form of either transcriptional regulation or transcription-independent reconstitution of enzymatic activity. This systematic, quantitative platform characterization provides a framework for engineering MESA to recognize novel ligands and for integrating these sensors into diverse mammalian synthetic biology applications.

Auristatin antibody drug conjugate physical instability and the role of drug payload.

The conjugation of hydrophobic cytotoxic agents such as monomethyl auristatin E (MMAE) to the interchain sulfhydryl groups of monoclonal antibodies (Mabs) through a protease-labile linker generates a heterogeneous drug load distribution. The conjugation process can generate high-drug-load species that can affect the physical stability of antibody-drug conjugates (ADCs). In this study, the mechanism of physical instability of ADCs was investigated by formulating the ADC pool as well as isolated drug load species in high and low ionic strength buffers to understand the effect of ionic strength on the stability of drug-conjugated Mabs. The results showed that the presence of high ionic strength buffer led to time-dependent aggregate and fragment formation of ADCs, predominantly ADCs with high-drug-load species under stress conditions. In addition, differential scanning calorimetry (DSC) results confirmed that there is a direct correlation between thermal unfolding and drug payload and that specific changes in the DSC thermogram profiles can be assigned to modifications by MMAE.