High-throughput T cell receptor engineering by functional screening identifies candidates with enhanced potency and specificity.

A major challenge in adoptive T cell immunotherapy is the discovery of natural T cell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human T cell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple in vitro and in vivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications.

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing.

Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

Leveraging Single-Cell Sequencing for Chimeric Antigen Receptor T Cell Therapies.

Chimeric antigen receptor (CAR)-T cell therapies against cancer continue to make inroads in the clinic. However, progress is still hindered by subpar efficacy against many tumors. Gaining a better understanding of CAR-induced T cell activation would help identify and remediate the causes of treatment failure. Increasingly, technologies to analyze the transcriptome are used to molecularly profile the behavior of CAR-T cells, both before and after treatment. Here, we describe recent work on how gene expression signatures, especially those obtained from single-cell RNA sequencing (scRNA-seq), can be used to characterize CAR design, production conditions, therapy combinations, and finally disease outcome. In the future, scRNA-seq could become a standard tool for the development and clinical monitoring of CAR-T cell therapies.