Precision targeting of autoantibody-producing IGHV1-69+ B cells in immune-mediated thrombotic thrombocytopenic purpura (iTTP) using chimeric antigen receptor T cells

Abstract Introduction : CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has shown promise for the treatment of B cell-mediated autoimmune diseases such as systemic lupus erythematosus (SLE), myositis, multiple sclerosis, and myasthenia gravis. However, CD19 is expressed in all B cells, leading to various degrees of immune suppression due to B cell aplasia and hypogammaglobulinemia. There is a dire need to develop more targeted CART therapies, especially in patients with non-malignant conditions. Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by autoantibodies targeting ADAMTS13 that neutralize its activity. This leads to accumulation of ultralarge von-Willebrand factor (UL-vWF) multimers that bind platelets and lead to microvasculature thromboses, which can cause ischemic organ injury. Remarkably, these anti-ADAMTS13 autoantibodies have been shown in >90% of patients to carry the immunoglobulin heavy-chain variable region IGHV1-69, highlighting its role in iTTP pathogenesis. Indeed, IGHV1-69 has been associated with antigen-independent signaling and is enriched across multiple B cell malignancies, including chronic lymphocytic leukemia and large B cell lymphoma, while composing only ~3% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV1-69 CAR T cells would be highly effective and safe against iTTP, as they would target IGHV1-69+ B cells responsible for the production of anti-ADAMTS13 autoantibodies while sparing normal B cells. Methods and Results :We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV1-69+ B-cell receptor (BcR) (CART1-69) using a single-chain variable fragment (scFv) derived from the humanized murine G6.3 monoclonal antibody. Using CRISPR/Cas9, we engineered multiple IGHV1-69- B-cell lines to express the IGHV1-69+ BcR (Mec1, Jeko1, Nalm6). In short-term cytotoxicity assays, we found that CD19-directed CAR T cells (CART19) exhibited strong cytotoxicity towards all B cell lines when compared to untransduced (UTD) control T cells (99% specific killing with CART19 against IGHV1-69+ and IGHV1-69-cells; p < 0.001 vs. UTD for both), while CART1-69 only showed significant cytotoxicity towards IGHV1-69-+ B cells (87.2% specific killing, p<0.001). In vivo, CART1-69 showed potent anti-tumor effects against an IGHV1-69+ tumor cell line and it expanded in the periphery to similar levels as CART19 (>200 CART/uL). To mimic iTTP-like disease in mice, we engineered the B-cell line Nalm6 to express and secrete an anti-ADAMTS13 patient-derived neutralizing antibody that is cross-reactive to both human and mouse ADAMTS13 (Nalm6-TTP). We injected 1x10^6 Nalm6-TTP cells on day 0, followed by 2.5x10^6 CAR+ T cells on day 4 (including UTD negative control cells, CART19 and CART1-69). To induce endothelial activation and microvascular thrombosis in the context of ADAMTS13 inhibition, on day 21 we collected blood and then intravenously injected mice with 1,000 IU/kg of human recombinant vWF + 200 pg/g of Shiga toxin, followed by another blood collection on day 22. Mice treated with UTD control T cells had a robust decrease in platelets following the vWF/Shiga toxin challenge (post-challenge/pre-challenge = -47%), while mice treated with either CART19 or CART1-69 had significantly smaller changes (CART19: -12%, p=0.004 vs. UTD; CART1-69: -15%, p=0.003 vs. UTD). Similarly, UTD-treated mice exhibited a significant decrease in hemoglobin following vWF/Shiga toxin challenge (-19%, p<0.001), while CART19 and CART1-69 showed no significant changes in hemoglobin (<2%). We then confirmed the specificity and safety of CART1-69 by co-culturing them with healthy donor B cells. While CART19 eliminated all B cells (specific killing 86%, p<0.001 vs. UTD), CART1-69 specifically depleted IGHV1-69+ B cells without causing B-cell aplasia (specific killing 4%, p=0.54). Conclusion :In summary, we introduce a novel paradigm for the treatment of autoimmune disease: the specific depletion of an entire family of immunoglobulin heavy-chain variable regions, IGHV1-69, while sparing the remainder of the B cell repertoire, for the treatment of iTTP, which is caused by IGHV1-69+ autoantibodies against ADAMTS13. We designed a novel CAR T cell product that is capable of specifically targeting IGHV1-69+ cells and is able to achieve similar control of iTTP-like symptoms in mice as CART19, without the long-term immune suppression commonly observed with CART19.