Design of diversified chimeric antigen receptors through rational module recombination.

Chimeric antigen receptor (CAR)-T cells have shown great promise in cancer therapy. However, the anti-tumor efficiency is limited due to the CAR-induced T cell apoptosis or exhaustion. The intracellular domain of CAR comprised of various signaling modules orchestrates CAR-T cell behaviors. The modularity of CAR signaling domain functions as the "mainboard" to assemble diversified downstream signaling components. Here, we implemented the modular recombination strategy to construct a library of CARs with synthetic co-signaling modules adopted from immunoglobin-like superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF). We quantitatively characterized the signaling behaviors of these recombinants by both NFAT and NF-κB reporter, and identified a set of new CARs with diverse signaling behaviors. Specifically, the 28(NM)-BB(MC) CAR-T cells exhibited improved cytotoxicity and T cell persistence. The synthetic approach can promote our understanding of the signaling principles of CAR molecule, and provide a powerful tool box for CAR-T cell engineering.

Design of Tunable Oscillatory Dynamics in a Synthetic NF-κB Signaling Circuit.

Although oscillatory circuits are prevalent in transcriptional regulation, it is unclear how a circuit's structure and the specific parameters that describe its components determine the shape of its oscillations. Here, we engineer a minimal, inducible human nuclear factor κB (NF-κB)-based system that is composed of NF-κB (RelA) and degradable inhibitor of NF-κB (IκBα), into the yeast, Saccharomyces cerevisiae. We define an oscillation's waveform quantitatively as a function of signal amplitude, rest time, rise time, and decay time; by systematically tuning RelA concentration, the strength of negative feedback, and the degradation rate of IκBα, we demonstrate that peak shape and frequency of oscillations can be controlled in vivo and predicted mathematically. In addition, we show that nested negative feedback loops can be employed to specifically tune the frequency of oscillations while leaving their peak shape unchanged. In total, this work establishes design principles that enable function-guided design of oscillatory signaling controllers in diverse synthetic biology applications.