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

Abstract

Chimeric antigen receptors (CARs) consist of an extracellular antigen-binding region fused to intracellular signaling domains, thus enabling customized T cell responses against target cells. Due to the low-throughput process of systematically designing and functionally testing CARs, only a small set of immune signaling domains have been thoroughly explored, despite their major role in T cell activation, effector function and persistence. Here, we present speedingCARs, an integrated method for engineering CAR T cells by signaling domain shuffling and functional screening by single-cell sequencing. Leveraging the inherent modularity of natural signaling domains, we generated a diverse library of 180 unique CAR variants, which were genomically integrated into primary human T cells by CRISPR-Cas9. Functional and pooled screening of the CAR T cell library was performed by co-culture with tumor cells, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), thus enabling high-throughput profiling of multi-dimensional cellular responses. This led to the discovery of several CAR variants that retained the ability to kill tumor cells, while also displaying diverse transcriptional signatures and T cell phenotypes. In summary, speedingCARs substantially expands and characterizes the signaling domain combinations suited for CAR design and supports the engineering of next-generation T cell therapies.

Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies

“…Advances in genome engineering and screening methods have offered numerous approaches to increase fitness of T cell therapies and overcome dysfunctional states. One approach is to tune CAR regulation and signaling itself by either targeting the CAR integration to place it under promoter regulation of the endogenous TCR alpha constant chain ( TRAC ) (Eyquem et al, 2017) or by screening a variety of different co-stimulatory CAR domains to identify CAR designs with favorable phenotypes (Di Roberto et al, 2021; Goodman et al, 2021; Kyung et al, 2021). A second approach uses CRISPR/Cas9 to ablate genes that restrict durable T cell function.…”