We developed a method in which the NS3 cis-protease from hepatitis C virus can be used as a ligand-inducible connection to control the function and localization of engineered proteins in mammalian cells. To demonstrate the versatility of this approach, we designed drug-sensitive transcription factors and transmembrane signaling proteins, the activities of which can be tightly and reversibly controlled through the use of clinically tested antiviral protease inhibitors.

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SUMMARYChimeric antigen receptor (CAR) T cell immunotherapy has the potential to revolutionize cancer medicine. However, excessive CAR activation, lack of tumor-specific surface markers, and antigen escape have limited the safety and efficacy of CAR T cell therapy. A multi-antigen targeting CAR system that is regulated by safe, clinically-approved pharmaceutical agents is urgently needed, yet only a few simple systems have been developed, and even fewer have been evaluated for efficacy in vivo. Here, we present NASCAR (NS3 ASsociated CAR), a collection of induc-ible ON and OFF switch CAR circuits engineered with a NS3 protease domain deriving from the Hepatitis C Virus (HCV). We establish their ability to regulate CAR activity using multiple FDA-approved antiviral protease inhibitors, including grazoprevir (GZV), both in vitro and in a xenograft tumor model. In addition, we have engineered several dual-gated NASCAR circuits, consisting of an AND logic gate CAR, universal ON-OFF CAR, and a switchboard CAR. These engineered receptors enhance control over T cell activity and tumor-targeting specificity. Together, our com-prehensive set of multiplex drug-gated CAR circuits represent a dynamic, tunable, and clinically-ready set of modules for enhancing the safety of CAR T cell therapy.

Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cis-protease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic/prokaryotic proteins and clinically approved inhibitors. Here we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3-peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, split-protein complementation. With our developed system, we discover a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.

Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cisprotease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic and non-prokaryotic proteins and clinically approved inhibitors. Here, we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, and split-protein complementation. With our developed system, we invented a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.