Temperature is a unique input signal that could be used by therapeutic agents, including engineered proteins and cells, to sense and respond to patient conditions or spatially targeted external triggers such as focused ultrasound. A critical capability for many envisioned applications is the ability to control the function of such therapies in situ to enable spatially and temporally specific activation at tumors and surrounding local tissue. However, among existing control methods, systemic chemical administration typically lacks the spatial precision needed to modulate activity at specific anatomic locations, while optical approaches suffer from poor light penetration into tissues. On the other hand, temperature can be controlled both globally and locally—at depth—using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of the patient’s condition. We have previously described the development of temperature-dependent protein actuators for control of gene expression in prokaryotic hosT-cells (1). Here, we generalize this technology to control protein-protein interactions with the aim of modulating arbitrary cell signaling pathways, including those of engineered therapeutic mammalian cells.To develop temperature-responsive protein-protein interaction domains, we turned to the thermo-sensitive bacterial transcriptional repressor TlpA. We demonstrated that this protein exhibits a tunable activation threshold which can be modulated within the biomedically relevant range of 32–46 °C, and that genes controlled by this repressor activate upon exposure to focused ultrasound hyperthermia applied noninvasively to a murine host (1). We then utilized a structure-based rational design strategy to convert the homodimeric TlpA protein into a pair of obligate heterodimers. We subsequently demonstrated that these heterodimers can be fused to sub-cellular anchoring moieties and cargo domains to conditionally sequester their payload into various compartments of human cells. Release of the cargo can be triggered via a mild thermal elevation, and the release temperature can be tuned via mutagenesis. In this study, we demonstrate that the temperature-sensing components of a bacterial transcriptional repressor can be isolated and repurposed into a generalized strategy to exert direct and rapid control over protein function in the context of both bacterial and mammalian cells. We aim to harness this technology to regulate therapeutically relevant processes in mammalian cells such as transcriptional activation and enzymatic signaling cascades. This class of bioswitches can be utilized to engineer a broad range of research tools and biological therapies with actuation driven by spatiotemporally precise noninvasively applied stimuli or by real-time sensing of host conditions.Reference: 1. Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro MG. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat Chem Biol 2016;13(1):75–80.

Citation Format: Dan Ilya Piraner, Mikhail G. Shapiro. Tunable thermal bioswitches for control of cell-based therapeutics [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B037.