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

项目总结/摘要 开发安全有效的嵌合抗原受体（CAR）转导的T细胞（CART）疗法， 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， 靶向肿瘤外毒性和CART细胞耗竭。到目前为止，还没有GBM特异性抗原， 均匀存在于所有GBM细胞上。另一方面，虽然非突变型GBM相关抗原（GAA）， 包括肝配蛋白A型受体2（EphA 2）和白细胞介素-13受体α2（IL-13 R α2），在人肝细胞中表达。 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GBM细胞的表达水平。 关注肿瘤外毒性。作为一种安全有效地靶向GAAs的方法，我们采用了一种新的合成 Notch“synNotch”受体系统，并开发了创新的“引发和杀死”T细胞电路。该系统 仅在GBM或CNS细胞上表达的第一抗原引发T细胞以诱导表达 识别IL-13 R α2和EphA 2的CAR，从而根除表达EphA 2或IL-13 R α 2的GBM细胞。 13Rα2。第一引发抗原应限制性地表达在CNS细胞上，但不表达在任何（非免疫）细胞上。 CNS）系统器官。那么该系统应该是安全的，因为在CNS内，EphA 2和IL-13 R α2是 仅在肿瘤细胞上表达，而在正常CNS细胞上不表达。我们发现了短蛋白聚糖（BCAN），一种定位于 神经元和神经胶质细胞表面，作为最有前途的引发抗原。当小鼠携带良好的 第9天，脑内GBM 6 PDX肿瘤接受单次IV输注α-BCAN工程化T细胞， 在synNotch α-EphA 2/IL-13 R α2 CAR（B-SYNC）电路中，所有小鼠均显示肿瘤完全消退 而不攻击CNS外的EphA 2/IL-13 R α2阳性细胞。此外，这些B-SYNC T细胞是 比常规的组成型表达的EphA 2/IL-13 R α2 CART细胞显著更有效， 与对照T细胞相比，具有上级CNS肿瘤归巢和较少耗竭的表型。我们将开发一个阶段 我的研究是为了评估我们的假设，即B-SYNC T细胞可以安全地作为单次静脉输注给药， B-SYNC T细胞能够浸润GBM组织，其中它们将被致敏， 表达针对EphA 2和IL-13 R α2的CAR。同时，我们将使用同系小鼠模型进行评估 我们的第二个假设是调节免疫调节机制可以进一步改善功能， B-SYNC T细胞的功效。我们将评估以下两个具体目标。 目标1.确定安全性以及归位和预充状态（即，CAR的表达）的IV输注 GBM患者中的B-SYNC T细胞。 目标2.评估潜在的耐药机制，并将缓解策略整合到B-SYNC方案中， 临床前同基因模型。在先前使用抗EGFRvIII CART细胞的试验中，CART细胞浸润GBM， 和诱导的免疫调节分子，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).