Novel anti-CD19 CAR-T cells for lupus nephritis treatment

项目摘要 狼疮性肾炎是系统性红斑狼疮（SLE）患者发病和死亡的主要原因。 SLE是一种典型的B细胞介导的自身免疫性疾病，目前仍难以治疗，约60%的狼疮患者将 最终发展为肾炎。活化或耗竭B细胞的方法为SLE提供了有吸引力的策略 疗法用抗B细胞表面标志物的单克隆抗体（如抗CD 20）消除B细胞 利妥昔单抗在类风湿性关节炎和多发性硬化症中显示出治疗前景，但不成功 在几个SLE的临床试验中抗CD 20抗体对B细胞的瞬时和不完全消耗可能 导致其未能取得令人满意的结果。因此，耗尽B细胞的新方法是 治疗难治性系统性红斑狼疮所需的药物最近，我们产生了一种重新工程化的抗CD 19 CAR（CD 19-CAR）。 BBz（86））衍生自经典的第二代CD 19-BBz（71）CAR。我们发现， CD 19-BBz（86）CAR-T细胞产生的细胞因子水平较低，增殖速度比经典CAR-T细胞慢。 CD 19-BBz（71）CAR T细胞，而它们保留了有效的细胞溶解活性。CD 19-BBz（86）CAR-T的临床试验 晚期淋巴瘤中的细胞表现出持久的抗肿瘤反应， 综合征（CRS）或神经毒性，代表淋巴瘤的安全和有效疗法（Ying Z等人，Nature Med25：947-953，2019）。重要的是，CD 19-BBz（86）CAR-T细胞治疗引起持续的B细胞耗竭， 淋巴瘤患者血清IgG和IgM水平的降低，这表明更安全的CD 19- BBz（86）CAR-T细胞也可用于治疗难治性SLE。在这项试点研究中，我们的目标是 开发一种安全持久的抗CD 19 CAR-T细胞疗法治疗狼疮性肾炎。本研究的假设 共表达细胞消融标志物tEGFR的新型再工程化CD 19-BBz（86）CAR-T细胞具有 安全且持久的细胞溶解活性，以持续的方式消耗CD 19 + B细胞并降低血清IG水平， 导致狼疮发病机制的持久缓解。本研究的具体目标是：目标1。测试 共表达细胞消融标志物tEGFR的再工程小鼠CD 19-BBz（86）CAR-T细胞的疗效 在小鼠SLE模型中消耗B细胞并减轻SLE发病机制。目标二。为了检测抗EGFR 抗体给药消耗共表达tEGFR的mCD 19-BBz（86）CAR-T细胞，以逆转 小鼠目标3。为了从机制上研究新的重新工程化的小鼠CD 19-T细胞的CAR触发的信号传导， BBz（86）CAR-T细胞与经典的第二代mCD 19-BBz（71）CAR-T细胞的体外比较。 这项拟议的研究是非常重要和新颖的，因为这项研究将导致开发一种新的，安全的， 以及用于狼疮性肾炎的长效抗CD 19 CAR-T细胞疗法。

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.