Project 3: Novel B-SYNC T cell therapy with CNS-specific expression of CAR as a safe and effective therapy for glioblastoma

项目摘要/摘要 安全有效的嵌合抗原受体(CAR)转导T细胞(CART)疗法的研究进展 胶质母细胞瘤(GBM)需要克服多重挑战，包括抗原表达的异质性，对 靶向肿瘤外毒性，并耗尽CART细胞。到目前为止，还没有GBM特异性抗原是 在所有的基底膜细胞上均有表达。另一方面，虽然非突变的GBM相关抗原(GaAs)， 包括肾上腺素A型受体2(EphA2)和白细胞介素13受体α2(IL-13Rα2)。 大多数GBM细胞，它们也在一些非中枢神经系统(CNS)器官中表达，提高了 对非肿瘤毒性的关注。作为一种安全有效地靶向砷化镓的方法，我们采用了一种新的合成方法 切入“synNotch”受体系统，并开发出创新的“启动和杀死”T细胞电路。在这个系统中， 第一个抗原只在GBM或CNS细胞上表达，它启动T细胞来诱导表达 识别IL-13Rα2和EphA2的CAR，从而根除表达EphA2或IL-2的基底膜细胞 13Rα2.第一个启动抗原应限制性地在中枢神经系统细胞上表达，而不是在任何(非 中枢神经系统)系统器官。那么系统应该是安全的，因为在中枢神经系统中，EphA2和IL-13Rα2是 仅在肿瘤细胞上表达，在正常中枢神经系统细胞上不表达。我们发现了短链蛋白聚糖(Bcan)，这是一种定位的蛋白多糖 对神经元和神经胶质细胞表面，作为最有希望的启动抗原。当小鼠发育良好时 第9天脑内GBM6PDX肿瘤接受α-bcan基因工程T细胞的单次静脉输注 SynNotchα-EphA2/IL-13Rα2 CAR(B-SYNC)电路，所有小鼠肿瘤完全消退 不攻击中枢神经系统外的EphA2/IL-13Rα-2阳性细胞。此外，这些B同步T细胞 明显比传统的、结构性表达EphA2/IL-13R的α2 CART细胞更有效， 与对照T细胞相比，中枢神经系统肿瘤归巢能力更强，耗尽表型更少。我们将制定一个阶段 我研究评估我们的假设，即B-sync T细胞可以作为一次静脉输注安全地应用于 患有GBM和B-sync T细胞的患者能够渗透到GBM组织，在那里他们将准备好 表达抗EphA2和IL-13Rα2的CAR。同时，利用同基因小鼠模型，我们将评估 我们的第二个假设是，免疫调节机制的调节可能进一步改善功能和 B-sync T细胞的疗效。我们将评估以下两个具体目标。 目的1.确定静脉输液的安全性以及归巢和启动状态(即CAR的表达) 银屑病患者B-sync T细胞的检测 目的2.评估潜在的耐药性机制，并将缓解策略纳入#年的B-SYNC方案 临床前同基因模型。在先前的抗EGFRvIII CART细胞试验中，CART细胞渗入了基底膜 诱导免疫调节分子，如程序性死亡配体1(PD-L1)。

Project Summary/Abstract The development of safe and effective chimeric antigen receptor (CAR)-transduced T cell (CART) therapy for glioblastoma (GBM) needs to overcome multiple challenges, including heterogeneity of antigen expression, on- target off-tumor toxicity, and exhaustion of CART cells. To date, there are no GBM-specific antigens that are uniformly present on all GBM cells. On the other hand, while non-mutant GBM-associated antigens (GAAs), including Ephrin type A receptor 2 (EphA2) and interleukin-13 receptor α2 (IL-13Rα2), are expressed in the majority of GBM cells, they are also expressed in some non-central nervous system (CNS) organs, raising the concern of 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 “prime-and-kill” T cell circuits. In this system, the first antigen, which is expressed exclusively on GBM or CNS cells, primes the T cells to induce the expression of a CAR that recognizes IL-13Rα2 and EphA2, thereby eradicating GBM cells expressing either EphA2 or IL- 13Rα2. The first priming antigen should be restrictedly expressed on CNS cells but not on cells of any (non- CNS) systemic organs. Then the system should be safe because, within the CNS, EphA2 and IL-13Rα2 are expressed only on tumor cells and not on normal CNS cells. We found Brevican (BCAN), a proteoglycan localized to the neuronal and glial cell surface, as the most promising priming antigen. When mice bearing well-established day 9 intracerebral GBM6 PDX tumors received a single IV infusion of T cells engineered with the α-BCAN synNotchα-EphA2/IL-13Rα2 CAR (B-SYNC) circuit, all mice demonstrated complete regression of tumor without attacking EphA2/IL-13Rα2-positive cells outside of CNS. Furthermore, these B-SYNC T cells were significantly more efficacious than conventional, constitutively expressed EphA2/IL-13Rα2 CART cells, with superior CNS tumor-homing and less exhausted phenotype compared to control T cells. We will develop a phase I study to evaluate our hypothesis that B-SYNC T cells can be safely administered as a single IV infusion in patients with GBM and that B-SYNC T cells are able to infiltrate the GBM tissue, wherein they will be primed to express the CAR against EphA2 and IL-13Rα2. Concurrently, using syngeneic mouse models, we will evaluate our 2nd hypothesis that modulation of immunoregulatory mechanisms may further improve the function and efficacy of B-SYNC T cells. We will evaluate the following two specific aims. Aim 1. Determine the safety as well as the homing and priming status (i.e., expression of CAR) of the IV-infused B-SYNC T cells in patients with GBM. Aim 2. Assess potential resistance mechanisms and integrate mitigation strategies into the B-SYNC regimen in preclinical syngeneic models. In a prior trial with anti-EGFRvIII CART cells, the CART cells infiltrated the GBM and induced immunoregulatory molecules, such as programmed death-ligand 1 (PD-L1).