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

Rocío Castellanos-Rueda, Raphaël B. Roberto, Florian Bieberich, Fabrice S. Schlatter, Darya Palianina, Oanh T. P. Nguyen, Edo Kapetanovic, Heinz Läubli, Andreas Hierlemann, Nina Khanna and Sai T. Reddy ()
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Rocío Castellanos-Rueda: ETH Zürich
Raphaël B. Roberto: ETH Zürich
Florian Bieberich: ETH Zürich
Fabrice S. Schlatter: ETH Zürich
Darya Palianina: University of Basel
Oanh T. P. Nguyen: ETH Zürich
Edo Kapetanovic: ETH Zürich
Heinz Läubli: University of Basel
Andreas Hierlemann: ETH Zürich
Nina Khanna: University of Basel
Sai T. Reddy: ETH Zürich

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-34141-8

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