Nonclinical safety assessment of SBT777101, a CAR-engineered regulatory T cell therapy for autoimmune diseases

Abstract Chimeric antigen receptor (CAR)-regulatory T cell (Treg) therapies represent a promising approach for autoimmune diseases, but their nonclinical safety assessment remains challenging due to antigen-, disease-, and species-specific pharmacology that limits the relevance of conventional animal models. SBT777101, a CAR-Treg therapy targeting citrullinated proteins associated with rheumatoid arthritis and hidradenitis suppurativa, was evaluated using a nonclinical strategy centered on in vitro and ex vivo New Approach Methodologies (NAMs) and in vivo studies benchmarked against untransduced polyclonal Tregs, which are manufactured identically but lack the CAR and have an established clinical safety record, complemented by immunodeficient mouse studies. We hypothesized that CAR engineering would not destabilize the Treg phenotype or introduce new toxicological liabilities relative to polyclonal Tregs. SBT777101 maintained phenotypic stability under repeated stimulation and proinflammatory conditions, produced minimal proinflammatory cytokines following activation, and demonstrated immunomodulatory activity comparable to untransduced Tregs. Tissue cross-reactivity and primary cell studies identified limited membrane-associated binding, no evidence of CAR-mediated cytotoxicity or activation in normal human tissues, and no off-target binding by broad protein array screening. Immunosuppressive activity toward natural killer and CD8+ T cell responses was limited and comparable to polyclonal Tregs. Lentiviral integration site analyses demonstrated polyclonal integration profiles without evidence of transformation or abnormal growth in a cytokine- and activation-independent growth assay. This study establishes a combined in vivo and NAM-based nonclinical safety framework for CAR-Treg therapies and demonstrates that CAR engineering of SBT777101 does not introduce new safety liabilities relative to unmodified polyclonal Tregs. These findings supported progression of SBT777101 into Phase 1 clinical trials and offer a generalizable approach for evaluating the safety of future CAR-Treg therapeutics.