The focus of this proposal is to develop and translate effective regulatory T cell (Treg) therapies to suppress anti-drug antibody (ADA) responses to biotherapeutics such as clotting factor replacement therapy for the monogenic disorder hemophilia. Despite high potential, current Treg-based treatments are hampered by low starting numbers, challenges in scalability, and unwanted off-target suppression. Redirecting specificity by engineering antibody-based synthetic receptors towards conformational antigenic epitopes can surmount these obstacles. Whereas this concept has been highly successful in generating chimeric antigen receptor (CAR) T cells for blood cancers, adapting engineered Tregs for tolerance applications is still evolving. This proposal derives from our recent findings that a synthetic T cell receptor fusion construct (TRuC) can suppress ADA responses to clotting factor VIII (FVIII) in a murine model of hemophilia. TRuCs combine the specificity of an antibody with the internal signaling machinery of a TCR by incorporating into the endogenous TCR-CD3 complex of the engineered Treg. We find that the TCR-like signaling of TRuC Tregs can deliver suppression in a physiological manner. In contrast, the non-physiological signaling of a CAR can negatively affect Treg suppressive capacity. Why different engineered receptors with the same scFv would result in dramatically divergent signaling and functional outcomes is as yet unclear. This proposal combines mechanistic understanding of synthetic receptor engineering with targeted potentiation of the Treg suppressive effect to achieve ADA tolerance. In Aim 1, we will test the hypothesis that careful considerations in synthetic receptor design, such as receptor affinity need to be made such that activation thresholds are regulated to maintain optimal suppressive function. Further, we will examine mechanisms by which engineered Tregs with specificity to a soluble antigen like FVIII can suppress antigen specific B cells. In Aim 2, we will explore strategies to enhance in vivo localization, persistence, and functionality of TRuC Tregs. We will co-administer a novel single chain IL-2 immunocytokine to drive selective proliferation of TRuC Tregs, biasing immune activation toward tolerogenic outcomes. In Aim 3, we will evaluate TRuC Treg efficacy in the presence of established ADAs in combination with pharmacological immunosuppressants to establish long term tolerance and lower the risk of ADA recurrence. Successful completion of these aims will generate potent antigen specific Tregs for effective suppression of ADA responses to biotherapeutics. These studies will provide proof of principle for the utility and mechanism of action of engineered Tregs and provide steps for clinical translation. The modularity of the components of this platform will allow ready extension to suppress ADAs for other conditions, in autoimmune disease and transplantation tolerance.