Precision targeting of immune-mediated thrombotic thrombocytopenic purpura (iTTP) with chimeric auto-antigen receptor (CAAR) T cells

Abstract Introduction: Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a life-threatening thrombotic microangiopathy characterized by thrombosis, thrombocytopenia, and anemia due to accumulation of ultralarge von-Willebrand factor (UL-vWF) multimers. Current standard-of-care therapy for acute iTTP episodes, which comprises corticosteroids, plasmapheresis and rituximab, induces remission in >90% of cases. However, approximately 15% of patients experience at least one relapse with many experiencing multiple relapses. Moreover, emerging evidence indicate that tissue damage likely accumulates even in the absence of clinically evident flares. Thus, a curative strategy that efficiently depletes ADAMTS13-specific B cell clones, is needed. Chimeric Antigen Receptor (CAR) T cell immunotherapy has shown promising initial results in a variety of B-cell mediated autoimmune disorders. However, pan-B cell targeting using CD19-, or CD20-directed T-cell immunotherapies lead to B-cell aplasia and hypogammaglobulinemia that can be prolonged and that have been associated with significant non-relapse mortality, which is particularly relevant for patients with non-malignant conditions. Therefore, we sought to develop an iTTP-specific engineered T cell platform that would selectively deplete autoantibody producing B cells while preserving protective B cells. Methods and Results: To develop a curative therapy for iTTP via selective depletion of anti-ADAMTS13 B cells, we targeted auto-antigen specific B-cell Receptors (BCRs). We generated Chimeric Auto-Antigen Receptors (CAARs) by employing combinations of ADAMTS13 domains as extracellular targeting domains, coupled to the 4-1BB-CD3zeta or CD28-CD3zeta signaling domains, to specifically engage anti-ADAMTS13 BCRs. ADAMTS13-CAAR T cells were manufactured following a typical ex vivo manufacturing paradigm including anti-CD3/CD28 activation beads followed by transduction using CAR-encoding lentiviral vectors and expansion over approximately 10 days. We first tested their efficacy and specificity using luciferase assays with Nalm6 target cells expressing BCRs derived from iTTP patients. ADAMTS13-CAAR T cells specifically recognized and killed B cells expressing multiple iTTP BCRs. A lead CAAR construct comprising the disintegrin, thrombospondin type 1, cysteine-rich, and spacer domains (DTCS) of ADAMTS13, was selected for further development, based on its expression level and in vitro cytotoxic activity. Using a xenograft model employing luciferase+ Nalm6 expressing an iTTP BCR (Clone 1 -303), we found that DTCS-28z and DTCS-BBz T cells effectively controlled target cell expansion compared to animals infused with non-transduced T cells (p< 0.05). Importantly, Nalm6 control by DTCS-CAR T cells was comparable to that conferred by anti-CD19 CAR T cells. Mice treated with either DTCS-based CAAR T cell showed no weight loss (p>0.999) or other signs of toxicity. We additionally performed co-culture assay with varying concentrations (up to 100 ug/ml) of patient-derived antibodies to determine if CAAR T cell cytotoxic function is affected in presence of soluble autoantibodies. Similarly, CAAR T cell function in the presence of soluble autoantigen i.e. ADAMTS13 was tested. Despite supra-physiological levels of either autoantibodies or autoantigen (> 1ug/ml ADAMTS13), DTCS-based CAARTs demonstrated efficient lysis of BCR expressing Nalm6 with minimal interference by competitive binding. The ability of ADAMTS13-CAARs to target polyclonal autoreactive patient B cells was indirectly assessed using a patient-plasma adsorption assay. DTCS-BBz T cells deplete patient antibodies compared to non-transduced T cells (p= 0.0153) with no impact on total IgG (p= 0.573). Hence, we demonstrated that the ADAMTS13 specific CAAR receptor design encompasses epitopes targeted by autoimmune B cell clones in iTTP patients. Conclusion: In summary, we have developed a novel ADAMTS13-based CAAR T platform that, in preclinical studies, selectively eliminates ADAMTS13 autoimmune B cells. This engineered T cell platform has the potential to be a curative therapy for patients with iTTP.