Enhancing CAR T-cell function through structural modifications and small-molecule drugs

Chimeric antigen receptor (CAR) based T-cell therapies are a novel therapeutic modality that has yielded remarkable results when treating refractory and relapsed patients with acute lymphoblastic leukemia, diffuse large cell B-cell lymphoma and multiple myeloma. However, relapses and refractory diseases still occur. Numerous approaches can be utilized to advance the CAR technology and circumvent the blockades hampering the CAR T-cell function. One keystone to improve CAR T-cell function is to assess the structural questions affecting CAR stability and interactions with off-target molecules. Commonly, the CARs function as dimers. However, the dimer-forming cysteine residues can cause unwanted interactions with other cysteine residues in the structure, causing oligomerization and fragmentation. The stability can be investigated by structural characterization of expressed CARs, including the analysis of the forms expressed. The other keystone, off-target interactions, can be studied by investigating the CAR-dependent activation via other cell types, followed by the characterization and fine-tuning of the CARs to evade such interactions. In this thesis, we have studied the CAR structure and functionality by fine-tuning the dimer-forming regions to evade impairing off-target interactions and to provide an on-target cancer cell-recognizing receptor activating the cytotoxic machinery upon antigen binding. We have developed a novel SIRPα-based spacer platform for CAR, enabling the modification to target different antigens. This modality is not restricted to T-cells and can be utilized in other therapies, including CAR NK-cell therapy. The other focus of the thesis was improving the CAR T-cell cytotoxic potential. In this work, we systemically screened over 500 small-molecule drugs and conducted a genome-scale CRISPR-Cas9 loss-of-function screening to identify suitable drugs and their mechanism to enhance CAR T cell cytotoxicity. In this study, we recognized various tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized acute lymphoblastic leukemia and diffuse large B-cell lymphoma B-cells to cytotoxic function by CAR T-cells. In summary, the CAR structure requires careful examination to remove unnecessary interactions hampering the overall function and efficacy. However, a stable structure that displays no off-target interactions might be insufficient for successful anti-tumor activity. Additional enhancement, e.g., via small-molecule drugs or other function-enhancing molecules, might be required for efficient tumor clearance.