A Novel Cotinine-Based System for Switchable Chimeric Antigen Receptor T Cell Immunotherapy

Autologous T cells engineered to express a chimeric antigen receptor (CAR-T cells) have shown impressive outcomes in hematologic malignancies. However, safety concerns derived from the inability to control CAR-T cells in vivo remain a significant challenge to expand the application of CAR-T cell therapy. Switchable CAR-T cells can allow for turning on and off the activation and cytotoxicity of CAR-T cells in vivo, potentially increasing the safety of next-generation CART products. To this goal, CAR constructs targeting non-tumor antigens, e.g., haptens and peptide tags, can be designed and combined with tumor-targeting units that are fused to the hapten or peptide tag. In this study, we developed a novel switchable CAR-T system using cotinine as the hapten and an anti-cotinine CAR-T cell product. Of note, cotinine is physiologically absent in human and pharmacologically inert. A mouse antibody to cotinine was developed and successfully humanized. The anti-cotinine CAR confirmed to be displayed on T-cell surface. We first tested this approach to target epidermal growth factor receptor 2 (HER2) in ovarian cancer. To target HER2, we developed an affibody-based antigen binding domain characterized by high stability, broad spectrum of specificity, and feasibility of large scale chemical synthesis. In in vitro cytotoxicity and cytokine release assays, the activity of CAR-T cells was controlled by cotinine-tagged anti-HER2 affibody in a HER2-specific and dose-dependent manner. We synthesized the cotinine-tagged HER2 affibodies using single isoform cotinine and selected the optimal cotinine-tagged anti-HER2 affibody at the cotinine-based switchable CAR-T system. In an orthotopic ovarian cancer model using NSG mice engrafted with SKOV3, tumor regression was observed only when CAR-T cells were administered in combination with the cotinine-tagged HER2 affibody. In subsequent studies, we demonstrated that the cotinine-based switchable CAR-T system can be applied to other tumor targets such as mesothelin and epidermal growth factor receptor (EGFR). Additional studies are ongoing to determine the kinetics of the on and off CAR-T activation of this system and define the optimal dosing strategy to ensure anti-tumor effect or to interrupt toxicity. In conclusion, we have developed a novel switchable cotinine-based CAR-T system to control CAR-T function in vivo and that could be broadly applicable to wide variety of tumors.