Development of a cellular therapy product with single specificity and improved persistence to prevent immunity to biotherapeutics

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Regulatory T cell (Treg) therapy is a highly promising approach for controlling unwanted or pathogenic immune responses in autoimmune disease, transplantation or in anti-drug antibody (ADA) formation following the administration of biotherapeutics such as clotting factor VIII (FVIII) replacement therapy in hemophilia A. Lack of specificity and limited persistence impedes clinical application of polyclonal Treg therapy, which can be surmounted by expressing antibody-based synthetic receptors such as a chimeric antigen receptor (CAR) or TCR fusion construct (TRuC) to redirect antigen specificity. TRuCs utilize the internal signaling machinery of a TCR by reconfiguring endogenous TCR-CD3 signaling to respond to antibody-based recognition. We recently showed in a preclinical hemophilia A model of FVIII deficiency that engineering antigen specificity by TRuC expressing Tregs delivers durable suppression of ADA responses by more faithfully mimicking the physiological TCR signaling of endogenous Tregs. However, dual recognition by both the synthetic receptor and endogenous TCR can increase the likelihood of off-target suppressive effects and lead to competition for extracellular expression and downstream signaling mediators. In this proposal, we will evaluate a novel synthetic hybrid receptor generated by replacing the α and β variable (V) regions of the TCR with the VH and VL domains of a FVIII specific antibody (FVIII V-swapTCR). A single-step CRISPR/Cas mediated integration of FVIII V-swapTCR into the TCRα constant (TRAC) locus should abolish endogenous TCR expression, thus eliminating dual antigen specificity, while allowing for more precise control of synthetic receptor expression. In Aim 1, we will establish the specificity, and functionality of TCR KO FVIII V- swapTCR Tregs. We will extensively characterize the phenotype and molecular pathways utilized by TCR KO FVIII V-swapTCR Tregs. Functional suppression by the single-specific engineered Tregs will be assessed in a model of ADA formation to FVIII replacement therapy in hemophilia A. In Aim 2, we will explore an innovative single chain immunocytokine to drive the selective proliferation of TCR KO FVIII V-swapTCR Tregs in vivo, thereby improving durability of suppression. The results of this study will provide pre-clinical evidence for effective suppression of ADA responses to biotherapeutics and enable rational therapeutic design and applicability to other immune disorders. Validating the principles of TCR KO Vswap TCR will provide proof of principle and will be a first step in engineering single-antigen specific “off the shelf” antigen specific TCR and MHCI KO FVIII V-swap Tregs to overcome alloreactive barriers, contributing to our long-term goal to generate universally applicable Tregs from healthy donors.