![]() A conventional CAR is rationally designed based on the fusion of modular gene elements: an extracellular antigen-recognition domain (frequently an antibody single chain variable fragment (scFv)), structural elements (a hinge, a transmembrane helix and peptide linkers), as well as one or more intracellular signal-transducing domains. A key observation from such studies is that the molecular components and design of a CAR have a major influence on the features of the transcriptional response 10. By going beyond methods for standard cell phenotyping (e.g., detection of surface markers by flow cytometry), transcriptional phenotyping can uncover gene expression patterns and metabolic pathways, or when used to track therapy progression and outcomes, can help to identify predictors of therapeutic efficacy. Recent advances in molecular immune profiling, such as single-cell sequencing and transcriptome analysis, are contributing important quantitative insights on CAR T cell-mediated responses and patient outcomes 9. While promising, all of these strategies require additional drug compounds or genetic modifications, introducing further complexity to a therapeutic regimen that is already laborious and sensitive. Furthermore, synthetic biology approaches that incorporate controllable domains 6, 7, 8 are being utilized to design CARs responsive to drugs or antigenic cues in order to tune the strength, duration and specificity of the inflammatory response. For example, co-administration with immune-checkpoint blockade inhibitors (ICB e.g., monoclonal antibodies targeting PD-1/PD-L1) can activate tumor-infiltrating T cells 4 or alternatively, armoring CAR T cells with additional genetic modifications to make them overexpress certain cytokines (e.g., IL-12, IL-23, etc.) can potentially sensitize tumors to cell-mediated cytotoxicity 5. In order to enhance CAR T cell responses against tumors, recent work has leveraged immunological mechanisms to counter the immunosuppressive microenvironment. Together, these pitfalls of CAR T cell therapies form substantial obstacles to their broader use against a wider range of cancer types. Furthermore, strong adverse events, such as cytokine release syndrome (CRS) and transient neurotoxicity 3 are frequently associated with treatment and thus represent considerable safety concerns. Instances of relapse, sometimes through antigen escape 2 have also highlighted the limitations of CAR T cell persistence and of the monoclonality of the infusion product. For example, solid tumor cancers, such as in the breast or lung, are more resistant to CAR T cell-mediated killing and have struggled to make progress clinically. However, success in this field has been difficult to replicate outside of hematological B cell malignancies. The infusion of autologous CAR T cells results in a cytotoxic response against tumor cells and, as in a classical immune reaction, this treatment can potentially result in persistent immunity, with CAR T cells recently observed in patients several years post-treatment 1. These treatments rely on synthetic protein receptors that have been engineered for precise molecular recognition of a cell surface antigen (e.g., CD19 on the surface of B cell lymphomas). 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.Ĭellular immunotherapies against cancer have made substantial progress in recent years, with six FDA-approved chimeric antigen receptor (CAR) T cell treatments against hematological malignancies. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. 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. 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. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. ![]()
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