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

• Published in: Nature communications Vol. 13; no. 1; pp. 6555 - 16
• Main Authors: Castellanos-Rueda, Rocío, Di Roberto, Raphaël B., Bieberich, Florian, Schlatter, Fabrice S., Palianina, Darya, Nguyen, Oanh T. P., Kapetanovic, Edo, Läubli, Heinz, Hierlemann, Andreas, Khanna, Nina, Reddy, Sai T.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 02.11.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 13/31; 38/39; 42/40; 49/47; 49/91; 631/337/2019; 631/61/338/552; 631/67/1059/2325; 631/67/580; Antigens; Automobiles; Cell activation; Cell culture; Chimeric antigen receptors; CRISPR; Engineering; Gene mapping; Gene sequencing; High-throughput screening; Humanities and Social Sciences; Humans; Immunological diseases; Immunotherapy; Intracellular signalling; Lymphocyte Activation; Lymphocytes; Lymphocytes T; Modularity; multidisciplinary; Neoplasms; Phenotypes; Receptors; Receptors, Antigen, T-Cell - genetics; Receptors, Chimeric Antigen - genetics; Science; Science (multidisciplinary); Screening; Signal Transduction; Signaling; T-Lymphocytes; Transcriptomics; Tumors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-34141-8

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. Chimeric antigen receptors (CAR) are a promising option for cell-based immunotherapy for cancer and other immune diseases. Here the authors develop speedingCARs, an integrated CAR design and screening platform based on modular signaling domain shuffling and single cell transcriptomic analyses, and test its potential for identifying and validating novel CAR designs.