Tuning Signaling in CAR T cells for Enhanced Therapeutic Efficacy

• Applicants: Professorin Dr. Judith Feucht; Professor Dr. Josef Leibold
• Subject Area: Immunology ; Hematology, Oncology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 554679299

Chimeric Antigen Receptor ("CAR") T cells have revolutionized the treatment of patients with hematological malignancies. For this approach, T cells are genetically engineered to express a CAR comprised of an antigen binding domain fused to costimulatory domain (e.g., 4-1BB ("BB" or CD28 ("28")) and the intracellular CD3z ("z") chain. CARs reprogram the metabolic and functional features of T cells and redirect their cytotoxic potential to a cell surface antigen expressed on target cells in an MHC independent manner. The success of CAR T cells in patients with hematological malignancies has led to the approval of several CAR T cell products by the FDA and the EMA. Still, there is a sizeable fraction of patients that do not respond or relapse after an initial response. Similarly, CAR T cell therapy has so far only shown limited results against solid tumors, which has been linked to the challenges immune cells are facing in a hostile cancer microenvironment. The signaling strength thereby plays a critical role for the functional and phenotypic characteristics of CAR T cells and is influenced by the co-stimulatory domain (e.g., 4-1BB vs. CD28) incorporated in the CAR design: 28z-based CARs are associated with high effector function and a more rapid T cell expansion relative to BBz-based CARs but are more prone to T cell exhaustion and demonstrate reduced persistence in vivo. These differences have been linked to distinct CAR signaling activities, with a central role of the PI3K/AKT signaling pathway. In this project, the Feucht and Leibold laboratories join forces to calibrate CAR T cell signaling activity with the goal to improve their functional persistence to prevent relapse in hematological malignancies and to enhance their efficacy against solid tumor cells. We propose to perform a CRISPR/Cas9 base editing screen in CAR T cells to introduce single nucleotide variants (SNVs) in PIK3CD or PIK3R1, thereby modulating PI3K/AKT signaling strength. Our rational builds on strong preliminary data uncovering an activating point mutant in PIK3CD as beneficial in the context of BBz CAR T cells. Noteworthy, due to the distinct CAR signaling activities and metabolic profiles of 28z and BBz based CAR constructs, we expect to identify different SNVs scoring in each of these settings. Hits from this screen will be followed up in established in vitro stress test assays and predictive immunocompetent and transplantation-based mouse models of B-cell malignancies, glioblastoma and gastric cancer. Our detailed analysis encompasses metabolic features, phenotype, and functional persistence of base-edited or wildtype CAR T cells as well as the impact of the tumor (immune) microenvironment on therapeutic potency of the CAR T cell product. By modulating PI3K/AKT signaling, we firmly believe to improve CAR T cell properties and we are confident that our interdisciplinary approach will yield important results and biological insights.

• DFG Programme: Research Grants