Development of novel synNotch CART cell therapy in adult patients with recurrent EGFRvIII+ glioblastoma

开发安全有效的（CAR）转导T细胞（CART）治疗胶质母细胞瘤（GBM）需要 克服多重挑战，包括靶向肿瘤外毒性、抗原表达的异质性，以及 耗尽CART细胞。在肿瘤中没有均匀存在的GBM特异性表面抗原， 组织.例如，突变型表皮生长因子受体（EGFRvIII）是GBM特异性的，但其表达是特异性的。 在肿瘤内是异质的。另一方面，虽然非突变的GBM相关抗原（GAA），如 如EphA 2和IL-13 R α2，在GBM中更均匀地表达，它们在GBM外的其他器官中的表达， 中枢神经系统（CNS）引起了对肿瘤外毒性的关注。作为一种安全有效地瞄准 GAAs，我们采用了一种新的合成Notch“synNotch”受体系统，并开发了创新的T细胞。 在该系统中，仅在脑或GBM细胞上表达的第一抗原（例如EGFRvIII）引发 T细胞诱导识别IL-13 R α2和EphA 2的CAR的表达，从而根除GBM细胞 表达EphA 2或IL-13α2。我们的数据显示，EGFRvIII-synNotch引发的IL-13 R α2/EphA 2 CAR是 作为GBM特异性信号，EGFRvIII有效但限制性激活，导致完全根除 具有异质性EGFRvIII表达但不攻击IL-13 R α2/EphA 2- CNS外的阳性细胞。此外，这些synNotch-CAR T细胞显著更有效， 比传统的组成型表达IL-13 R α2/EphA 2 CAR T细胞， 持久性（体内>100天）。总之，我们的数据表明，synNotch CAR T细胞可以彻底改变 通过克服肿瘤外毒性、抗原异质性和缺乏免疫原性， 坚持不懈我们的目标是建立良好生产规范的发展过程 EGFRvIII引发的IL-13 R α2/EphA 2 CAR T细胞的产物，以获得研究性新药（IND） 批准，并开发和进行I期临床试验的患者复发性GBM。在目标1中，我们 生产GMP级慢病毒载体，并建立GMP级慢病毒载体生产的标准程序。 EGFRvIII引发的IL-13 R α2/EphA 2 CAR T细胞。在目标2中，我们将完成体外和体内研究， 体外研究包括确认抗原特异性引发，特异性 细胞毒性和不存在可复制慢病毒。在体内，我们将确认临床前疗效， 毒理学、组织生物分布和植入以及synNotch CART细胞的持久性。在目标3中， 作为第一个人体1期临床试验，我们将确定synNotch CART细胞在 复发性EGFRvIII+ GBM患者。在随后的扩展队列中，在复发性EGFRvIII+的患者中， GBM，我们将在手术前静脉内输注synNotch CART细胞，然后评估浸润， 和移植细胞在切除的GBM中的功能。这些研究将使我们能够确定最佳剂量 不仅基于耐受性，还基于药效学评估。

Development of safe and effective (CAR)-transduced T cell (CART) therapy for glioblastoma (GBM) needs to overcome multiple challenges, including on-target off-tumor toxicity, heterogeneity of antigen expression, and exhaustion of CART cells. There are no GBM-specific surface antigens that are uniformly present in tumor tissue. Mutant epidermal growth factor receptor (EGFRvIII), for example, is GBM-specific but its expression is heterogenous within the tumor. On the other hand, while non-mutant GBM-associated antigens (GAAs), such as EphA2 and IL-13Rα2, are more uniformly expressed in GBM, their expression in other organs outside the central nervous system (CNS) raises concern for off-tumor toxicity. As a way to safely and effectively target GAAs, we have adopted a novel synthetic Notch “synNotch” receptor system and developed innovative T cells. In this system, the first antigen, which is expressed exclusively on brain or GBM cells (e.g. EGFRvIII), primes the T cells to induce expression of a CAR that recognizes IL-13Rα2 and EphA2, thereby eradicating GBM cells expressing either EphA2 or IL-13α2. Our data show that EGFRvIII-synNotch primed IL-13Rα2/EphA2 CAR are effectively but restrictedly activated by EGFRvIII as the GBM-specific signal, leading to complete eradication of patient-derived xenografts with heterogeneous EGFRvIII expression but without attacking IL-13Rα2/EphA2- positive cells outside of CNS. Furthermore, these synNotch-CAR T cells were significantly more efficacious than conventional, constitutively expressed IL-13Rα2/EphA2 CAR T cells, and were associated with excellent persistence (>100 days in vivo). Taken together, our data indicate that synNotch CAR T cells can revolutionize the CAR T therapy for solid cancers by overcoming the off-tumor toxicity, antigen heterogeneity and lack of persistence. Our goals are to establish the process for development of Good Manufacturing Practice products of EGFRvIII-primed IL-13Rα2/EphA2 CAR T cells, to obtain an Investigational New Drug (IND) approval, and to develop and conduct a phase I trial in patients with recurrent GBM. In Aim 1, we will generate GMP-grade lentiviral vector and establish standard procedures for GMP-grade manufacturing of the EGFRvIII-primed IL-13Rα2/ EphA2 CAR T cells. In Aim 2, we will complete both in vitro and in vivo studies that are required for submission of an IND. In vitro studies include confirmation of antigen-specific priming, specific cytotoxicity, and absence of replication competent lentivirus. In vivo, we will confirm preclinical efficacy, toxicology, tissue biodistribution, and engraftment as well as persistence of synNotch CART cells. In Aim 3, in a first-in-human phase 1 clinical trial, we will determine safety and toxicity of the synNotch CART cells in patients with recurrent EGFRvIII+ GBM. In a subsequent expansion cohort, in patients with recurrent EGFRvIII+ GBM, we will infuse synNotch CART cells intravenously prior to surgery, and then evaluate infiltration, priming and function of the infused cells in the resected GBM. These studies will allow us to determine optimal dosing based not only on tolerability but also on pharmacodynamic assessments.