Project Summary Lupus nephritis is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). SLE, a classic B cell-mediated autoimmune disease, is still difficult to treat and about 60% of lupus patients will eventually develop nephritis. Approaches that inactivate or deplete B cells offer attractive strategies for SLE therapy. B cell depletion with a monoclonal antibody against the B cell surface marker, such as anti-CD20 Rituximab, has shown therapeutic promise in rheumatoid arthritis and multiple sclerosis, but was unsuccessful in several clinical trials for SLE. Transient and incomplete nature of B cell depletion by anti-CD20 antibodies may have contributed to its failure to achieve satisfactory outcomes. Hence, new approaches to deplete B cells are needed for the treatment of refractory SLE. Recently, we generated a re-engineered anti-CD19 CAR (CD19- BBz(86)) derived from the classic second-generation CD19-BBz(71) CAR. We found that the re-engineered CD19-BBz(86) CAR-T cells produced lower levels of cytokines and proliferated at a slower rate than the classic CD19-BBz(71) CAR T cells, while they retained potent cytolytic activity. A clinical trial of CD19-BBz(86) CAR-T cells in advanced-stage lymphomas showed durable antitumor responses without causing cytokine release syndrome (CRS) or neurotoxicity, representing a safe and potent therapy for lymphoma (Ying Z et al. Nature Med 25: 947-953, 2019). Importantly, CD19-BBz(86) CAR-T cell therapy caused sustained B cell depletion and reduction of serum IgG and IgM levels in the patients with lymphoma, which suggests that the safer CD19- BBz(86) CAR-T cells could be used for the treatment of refractory SLE as well. In this pilot study, we aim to develop a safe and long-lasting anti-CD19 CAR-T cell therapy for lupus nephritis. The hypothesis of this study is that the novel re-engineered CD19-BBz(86) CAR-T cells co-expressing a cell ablation marker tEGFR have a safe and long-lasting cytolytic activity to deplete CD19+ B cells and reduce serum Ig levels in a sustained manner, resulting in the lasting alleviation of lupus pathogenesis. The specific aims of this study are: Aim 1. To test the efficacy of the re-engineered mouse CD19-BBz(86) CAR-T cells that co-express a cell ablation marker tEGFR to deplete B-cells and alleviate SLE pathogenesis in a mouse SLE model. Aim 2. To test whether anti-EGFR antibody administration depletes mCD19-BBz(86) CAR-T cells coexpressing tEGFR to reverse B cell aplasia in mice. Aim 3. To mechanistically investigate the CAR-triggered signaling of the novel re-engineered mouse CD19- BBz(86) CAR-T cells in comparison with the classic second-generation mCD19-BBz(71) CAR-T cells in vitro. This proposed study is highly significant and novel, since this study will lead to the development of a novel, safe and long-lasting anti-CD19 CAR-T cell therapy for lupus nephritis.