Engineering chimeric antigen receptor signaling architectures for T cell immunotherapies
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Author
Date
2024Type
- Doctoral Thesis
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Abstract
Chimeric Antigen Receptors (CARs) are rationally designed synthetic receptors that are engineered to redirect the specificity and effector function of T lymphocytes toward a target surface antigen. Particularly in the realm of cancer therapeutics, CAR T cells designed to target tumor-associated antigens have emerged as a groundbreaking approach within cellular immunotherapy, showcasing remarkable clinical efficacy in the treatment of B cell malignancies. The field of CAR T cell therapy offers a promising avenue for cancer treatment, yet significant hurdles remain in optimizing efficacy and applicability across diverse clinical contexts. This emphasizes the need for innovative tools to identify the factors impeding the success of current CAR T cell therapies and to drive the development of enhanced CAR designs. Through the combination of directed evolution approaches and single-cell sequencing techniques, we contribute to this effort by establishing a tool for efficient high-throughput diversification and screening of CAR signaling architectures, thereby accelerating the engineering of more effective CAR constructs and advancing our comprehension of CAR-induced T cell phenotypes.
Chapter 1 serves as an introduction to CAR T cell therapy, emphasizing the emerging role of single-cell RNA sequencing (scRNAseq) in characterizing T cell heterogeneity and CAR-induced phenotypes. Whether in preclinical settings or during clinical trials, scRNAseq is rapidly becoming an essential tool for studying CAR-T cell behavior. This proves particularly valuable to assess the therapeutic potential of new engineering strategies or receptor candidates, as well as providing insights into the molecular profiles associated with disease outcomes. Furthermore, this chapter delves into the principles of traditional and high-throughput CAR T cell engineering, representing the current frontier in addressing challenges within CAR T cell therapies and laying the groundwork for subsequent Chapters.
In Chapter 2, we introduce speedingCARs, a method for effective CAR T cell engineering that integrates CRISPR Cas9 genome editing, pooled functional assays and single-cell sequencing to perform high-throughput screening of CAR signaling domain libraries. Despite the critical role of CAR signaling domains in T cell activation, only a limited number of immune signaling architectures have been explored. By leveraging the modularity of signaling proteins and the extensive existing diversity of immune receptor domains involved in the T cell signaling network, we generate a library of 180 unique CAR variants through domain recombination, driving diversification of CAR signaling. After targeted genomic integration of the CAR library into primary human T cells, in vitro tumor cell co-culture, followed by scRNAseq, enables high-throughput functional screening of CAR-induced phenotypes. This multidimensional readout provides valuable insights into the phenotypic diversity triggered by different CAR architectures and allows the identification of CAR variants driving enhanced antitumor effector properties. This comprehensive approach can be used to expand the CAR signaling domain combination space and guide the selection of new variants better suited to tackle current immunotherapeutic challenges.
In Chapter 3 the speedingCARs workflow is adapted to systematically investigate the impact of varying the choice, number and order of 5 costimulatory domains on T cell phenotype. Additionally, we introduce an in vitro model of CAR T cell dysfunction, simulating chronic tumor stimulation, a recurring limitation during clinical treatment. Using a mid-sized library of 32 candidates, we use single-cell sequencing to dissect the intricate relationship between CAR design and T cell phenotype during activation and long-term persistence. Parallel comparisons of CAR variants at early, middle and late time points during chronic stimulation reveal the predominant influence of membrane-proximal domains in driving T cell phenotype, with CD40 costimulation emerging as crucial for promoting potent and persistent T cell responses. These findings not only deepen our understanding of CAR T cell biology but also offer actionable insights for refining CAR design strategies to enhance therapeutic efficacy.
The culmination of these chapters underscores the effectiveness of integrating high-throughput CAR engineering strategies with single-cell transcriptomics to unravel the complex relationship between CAR signaling and T cell phenotype. Moreover, they emphasize the value of employing domain recombination, harnessing naturally optimized immune receptor domains, to expand CAR signaling architectures that rewire T cell signaling. By providing a highly efficient means of screening libraries of synthetic receptors, this thesis lays the foundation for the advancement of more effective CAR T cell therapies. Furthermore, this integrated approach holds great promise for unlocking the full therapeutic potential of CAR T cell therapy and enhancing outcomes for cancer patients. Show more
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https://doi.org/10.3929/ethz-b-000695219Publication status
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ETH ZurichOrganisational unit
03952 - Reddy, Sai / Reddy, Sai
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