Enhancing iPSC-derived off-the-shelf CAR T cells for applications in cancer

• Applicant: Dr. Roland Windisch
• Subject Area: Hematology, Oncology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548112675

The proposal addresses the current limitation in the production of chimeric antigen receptor (CAR) T cells from induced pluripotent stem cells (iPSCs), which predominantly yield cytotoxic CD8+ T cells. Emerging evidence highlights the importance of CD4+ T cells in anti-tumor immunity, indicating the need for optimal CD4/CD8 ratios in off-the-shelf CAR T cells. In this proposal, different strategies to enhance CD4+ T cell differentiation from iPSCs will be investigated. The research is structured into six work packages (WPs). WP1 focuses on identifying key candidate genes influencing CD4+ T cell differentiation using genetic screening methods. This involves compiling a list of genes associated with positive modulation of lymphoid differentiation, followed by iPSC differentiation and gene modulation using shRNA and CRISPR technologies during T cell specification. In contrast to genetic screens, WP2 involves evaluating the impact of small molecules on CD4+ T cell differentiation. Screening occurs at key differentiation stages using an epigenetics screening library. Additionally, a library of small molecules beyond epigenetic regulators, affecting various aspects of cell biology, will be compiled based on a literature review. WP3 aims to enhance the generation of CD4+ T cells from iPSCs without directly manipulating target genes or using small molecules. The aim is to increase CD4+ T cell generation by introducing MHC class II-expressing cells, particularly dendritic cells, during differentiation. Two approaches are employed in WP3. First, additional cytokines will be introduced to promote the emergence of DCs while maintaining proT cell differentiation favoring CD4+ T cell derivation. Second, MHC class II-expressing cells, e.g., artificial antigen-presenting cells, will be introduced during T cell differentiation from iPSCs. In the next step (WP4) results from WP1 and WP2 will be integrated and analyzed to select key genes promoting CD4+ T cells. The top candidates undergo further investigation, including differentiation assays and detailed phenotyping through RNA-seq and single-cell RNA-seq. The objective of WP5 is to assess the potential use of iPSC-derived T cells, generated through target gene modulation or co-culture with MHC class II expressing cells, for the production of CAR T cells. After transduction with a CD19-CAR lentiviral vector cytotoxicity and cytokine secretion profiles will be evaluated. Finally, in WP6 the antitumor activity of the manufactured CAR T cells will be determined in a tumor xenograft model using NSG mice injected with CD19+ OCI-Ly1 tumor cells. CAR T cell functionality is assessed by monitoring tumor burden and persistence over time. Overall, the study aims to optimize iPSC-derived CAR T cell manufacturing processes for enhanced therapeutic applications by addressing the challenge of generating a balanced CD4/CD8 T cell ratio with a focus on CD4+ T cells’ beneficial properties in anti-tumor immunity.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor George Q. Daley, Ph.D.