Establishing reverse chimeric antigen receptor T cells for precise targeting of immunemediated thrombotic thrombocytopenic purpura

Immune-mediated Thrombotic Thrombocytopenic Purpura (iTTP) is a life-threatening autoimmune disorder caused by autoantibodies that target the metalloprotease ADAMTS13. This prevents the cleavage of Ultra-Large von Willebrand Factor (UL-VWF), thereby forming microthrombi in the microvasculature. While current treatments are effective, ADAMTS13 relapses occur in up to 15% of the patients after rituximab administration. In this study, we propose a novel approach addressing the unmet need for treatment of patients with ADAMTS13 refractoriness and for patients requiring repeated treatment. Our strategy involves developing two targeted therapies that aim to achieve highly effective and safe B cell depletion. For the first time, we employ adapter Chimeric Antigen Receptor (CAR) T cells called Reverse CAR (RevCAR) T cells to (i) achieve a broader and deeper B cell depletion by targeting CD19 and (ii) selectively deplete the disease-causing anti-ADAMTS13 autoreactive B cells to increase precision and safety in iTTP treatment. Therefore, we have developed novel Target Modules (RevTMs) against CD19+ and anti-ADAMTS13 autoreactive B cells. We show that the RevCAR T cells successfully and specifically kill target hybridomas and Nalm-6 cells in a RevTM-dependent manner and at low effector-to-target cell ratios. Moreover, the RevCAR T cells are able to eliminate the target cells even in the presence of high concentrations of ADAMTS13 autoantibodies. Overall, our findings demonstrate for the first time, a proof-ofconcept that anti-ADAMTS13 autoreactive B cells can be selectively depleted by the RevCAR T cell platform paving the way for an effective and safe therapy for iTTP.