Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

Small molecule-inducible gene regulatory systems in mammalian cells: progress and design principles.

Small molecule-inducible gene circuits are some of the most important tools in biology because they provide a convenient way to exert precise regulation of biological systems. These systems typically are designed to govern gene activation, repression, or disruption at multiple levels, such as through genome modification, transcription, translation, or post-translational regulation of protein activity. Due to their importance, many new systems have been created in the past few years to address different needs or afford orthogonality. They can be broadly characterized based on the inducer used, the mode of regulation, and the effector protein enabling the regulation. Furthermore, each synthetic circuit has varying performance metrics and design considerations. Here, we provide a concise comparison of recently developed tools and recommend standardized metrics for evaluating their performance and potential as biological interrogators or therapeutics.

Ganglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse.

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be an effective strategy against hematological malignancies but persistence and activity against solid tumors must be further improved. One emerging strategy for enhancing efficacy is based on directing CAR T cells to antigen presenting cells (APCs). Activation of CAR T cells at the immunological synapse (IS) formed between APC and T cell is thought to promote strong, persistent antigen-specific T cell-mediated immune responses but requires integration of CAR ligands into the APC/T-cell interface. Here, we demonstrate that CAR ligand functionalized, lipid-coated, biodegradable polymer nanoparticles (NPs) that contain the ganglioside GM3 (GM3-NPs) bind to CD169 (Siglec-1)-expressing APCs and localize to the cell contact site between APCs and CAR T cells upon initiation of cell conjugates. The CD169+ APC/CAR T-cell interface is characterized by a strong optical colocalization of GM3-NPs and CARs, enrichment of F-actin, and recruitment of ZAP-70, indicative of integration of GM3-NPs into a functional IS. Ligands associated with GM3-NPs localized to the APC/T-cell contact site remain accessible to CARs and result in robust T-cell activation. Overall, this work identifies GM3-NPs as a potential antigen delivery platform for active targeting of CD169 expressing APCs and enhancement of CAR T-cell activation at the NP-containing IS.