Degron-based bioPROTACs for controlling signaling in CAR T cells.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in pre-clinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous, cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit is able to disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are a powerful tool for expanding the cell engineering toolbox for CAR T cells.

De novo-designed minibinders expand the synthetic biology sensing repertoire.

Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled "smart" therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.

Intravitreal vascular endothelial growth factors hypertension, proteinuria, and renal injury: a concise review.

PURPOSE OF REVIEW: Nearly 20 years ago, vascular endothelial growth factor (VEGF)inhibitors (VEGFi) were adapted from systemic use from antiangiogenesis roles to intravitreal uses. Initially bevacizumab a murine immunoglobulin was injected 'off label' as a treatment for diabetic macular edema and age-related macular degeneration. Throughout the following decade aflibercept and finally ranibizumab were adapted and obtained Food and Drug Administration approval for intravitreal use. Initially systemic absorption was thought to be quite low after intravitreal injections and was quoted as being 200-fold lower than levels postulated to induce significant VEGF inhibition. Pharmacodynamic studies obtained in 2014 and again in 2017 revealed significant systemic absorption and detectable VEGF inhibition, this has since been confirmed in multiple subsequent studies. RECENT FINDINGS: A few case reports of renal dysfunction and glomerular disease related to VEGFi were initially identified. Mixed findings on effects on blood pressure were noted in studies. More recently, 32 cases of de-novo glomerular disease and/or proteinuria exacerbation were identified. New studies have corroborated increased blood pressure, proteinuria exacerbation in patients with pre-existing nephrotic syndrome, and systemic VEGF depletion. Further, the most common lesion of systemic VEGFi nephrotoxicity, thrombotic microangiopathy, has recently been reported by our group. SUMMARY: We will review the pharmacokinetic, translational, and epidemiological data that year upon year establish the finite-yet real risk of intravitreal VEGFi.

Programmable protein circuits in living cells.

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications.