A 3-In-One Design of CAR Tregs for Robust Immunotolerance - PROJECT SUMMARY Regulatory T cells (Tregs) are essential for maintaining immunotolerance, protecting tissues from immune- mediated damage while preserving normal immune function. Chimeric Antigen Receptor T regulatory cells (CAR Tregs) have emerged as a cutting-edge therapeutic approach due to their enhanced specificity, potency, and reliable sourcing. However, despite their promise, current CAR Tregs have struggled to achieve durable tolerance in clinical applications, exposing critical gaps in efficacy and highlighting the need for innovative strategies to improve their therapeutic potential. This project introduces a transformative 3-In-One CAR Treg design to address these limitations. Unlike conventional CAR Tregs, which rely on disease-specific antigens for activation and suppression, the 3-In-One approach utilizes effector T cells (eTconv) as a multi-functional target. In this design, eTconv serve as the attractant, activator, and suppression focus of CAR Tregs, thereby streamlining their activation and enhancing their suppressive efficacy. This strategy was crafted based on core working mechanisms of CAR Tregs that center around eTconv rather than antigens and is expected to significantly improve the efficiency and durability of CAR Treg-mediated immunotolerance. We propose using Programmed Death-1 (PD-1), a marker predominantly expressed on activated eTconv, as the target for this innovative CAR Treg design. PD-1CAR Tregs are expected to exhibit superior suppressive efficacy compared to traditional antigen-specific CAR Tregs by selectively suppressing eTconv without affecting naïve T cells or normal immune responses. This targeted approach offers a significant advantage over conventional CAR Tregs, whose application is often restricted to specific antigen-driven diseases. In this R21 project, we will develop and evaluate PD-1CAR Tregs with varied affinities for PD-1, alongside a reference MOGCAR Treg that targets a specific myelin antigen. Our research aims to: I. Characterize PD-1CAR Tregs. We will assess binding specificity, regulatory phenotypes, cytokine secretion, suppression of eTconv, and bystander immunosuppressive effects. II. Evaluate Therapeutic Efficacy and Safety. Using two experimental autoimmune encephalomyelitis (EAE) models representing distinct autoimmune conditions, we will measure the ability of PD-1CAR Tregs to restore immunotolerance. Outcomes will include clinical EAE scores, suppression of demyelinating eTconv activity. A critical advantage of PD-1CAR Tregs is their potential applicability across multiple antigen-driven diseases, avoiding the requirement to develop different CAR Tregs for different medical conditions. Furthermore, we will rigorously evaluate whether the immunotolerance induced by PD-1CAR Tregs selectively targets pathogenic responses without impairing host immunity to infections. In summary, the 3-In- One design represents a paradigm shift in CAR Treg therapy, combining efficiency, specificity, and versatility. By targeting eTconv via PD-1, this approach promises to revolutionize CAR Treg-based treatments for autoimmune diseases and other conditions requiring immune tolerance.
