Genetic Approaches to Optimize CAR T cells for Glioblastoma Therapy

胶质母细胞瘤（GBM）是最常见和最具侵袭性的原发性脑肿瘤， 无法治愈因此，迫切需要开发新的治疗方法。基因修饰的T细胞表达 嵌合抗原受体（汽车）具有作为特异性靶向细胞毒性工具的潜力， GBM。CAR T细胞疗法已成功用于血液恶性肿瘤，但面临多重挑战 需要多方面的方法使CAR T细胞成功用于GBM。研究 为此，我们开发了一种针对GBM相关肿瘤IL 13 R α2的单链可变区片段（scFv 抗原，并产生了IL 13 Ra 2-CAR。IL 13 R α2-CAR T细胞仅识别IL 13 R α2阳性胶质瘤 细胞，并在临床前异种移植和免疫活性动物模型中具有抗胶质瘤活性。然而，在这方面， 肿瘤最终复发，与人类的情况相似。治疗失败的主要原因包括（i） CAR T细胞不能在免疫抑制性肿瘤环境中持续存在，（ii）抗原丢失变体 当靶向单一抗原时，和（iii）由于CAR T细胞不能有效地运输到肿瘤部位， 肿瘤产生的趋化因子与CAR T细胞表达的趋化因子受体之间的错配。在 机制研究，我们已经证明了有限的IL 13 R α2-CAR T细胞持久性和发展， 抗原丢失变体。此外，我们在异种移植模型中显示，CAR T细胞中IL 15的转基因表达， 细胞增强其持久性和抗神经胶质瘤活性。然而，这些异种移植研究是有限的;目标是 本R 01的目的是在免疫活性动物模型中进行机制研究，并评估遗传 增强IL 13 R α2-CAR T细胞的抗胶质瘤活性的方法。因此，我们现在假设， IL 13 R α2-CAR T细胞可以进一步遗传工程化以优化其抗GBM活性， 增强它们的持久性，靶向多种肿瘤抗原，并改善它们向肿瘤的运输， 网站.目的1研究表达IL 15的CAR T细胞是否可以抵抗免疫抑制性肿瘤 环境中的同源GBM模型。目的2优化CAR T细胞以靶向IL 13 R α2和EphA 2， 胶质瘤相关抗原。目的3研究CAR T细胞向GBM的运输是否可以通过以下方式改善： CCR 2的转基因表达，CCR 2是一种识别CCL 2的趋化因子受体，CCL 2是一种由GBM产生的趋化因子。 在拨款结束时，我们将解决GBM CAR T细胞治疗的三个主要障碍。而 我们将使用我们的数据来证明未来利用优化的IL 13 R α2-CAR T细胞进行临床研究的合理性 对于GBM患者，我们改良的T细胞治疗方法应适用于广泛的实体瘤。 肿瘤的

Glioblastoma (GBM), the most frequently occurring and aggressive primary brain tumor, remains virtually incurable. Thus, there is an urgent need to develop new therapies. Genetically modified T cells expressing chimeric antigen receptors (CARs) have the potential to serve as a unique cytotoxic tool to specifically target GBM. CAR T cell therapy has been successful for hematological malignancies, but multiple challenges posed by the brain tumor environment require a multifaceted approach for CAR T cells to succeed for GBM. To study this, we have developed a single-chain variable fragment (scFv) specific for IL13Rα2, a GBM-associated tumor antigen, and have generated an IL13Rα2-CAR. IL13Rα2-CAR T cells only recognize IL13Rα2-positive glioma cells and had anti-glioma activity in preclinical xenograft and immune-competent animal models. However, tumors eventually recurred, paralleling the situation in humans. Major causes of treatment failure include (i) the inability of CAR T cells to persist within an immunosuppressive tumor environment, (ii) antigen-loss variants when a single antigen is targeted, and (iii) the inability of CAR T cells to efficiently traffic to tumor sites due to a mismatch between chemokines produced by the tumor and chemokine receptors expressed by CAR T cells. In mechanistic studies, we have demonstrated limited IL13Rα2-CAR T cell persistence and the development of antigen-loss variants. In addition, we showed in xenograft models that transgenic expression of IL15 in CAR T cells enhances their persistence and anti-glioma activity. However, these xenograft studies are limited; the goal of this R01 is to perform mechanistic studies in immune-competent animal models and evaluate genetic approaches to enhance the anti-glioma activity of IL13Rα2-CAR T cells. Thus, we now hypothesize that IL13Rα2-CAR T cells can be further genetically engineered to optimize their anti-GBM activity by enhancing their persistence, targeting multiple tumor antigens, and improving their trafficking to tumor sites. Aim 1 investigates whether IL15-expressing CAR T cells can resist the immunosuppressive tumor environment in syngeneic GBM models. Aim 2 optimizes CAR T cells to target both IL13Rα2 and EphA2, two glioma-associated antigens. Aim 3 investigates if trafficking of CAR T cells to GBMs can be improved by the transgenic expression of CCR2, a chemokine receptor that recognizes CCL2, a chemokine produced by GBMs. At the conclusion of the grant, we will have addressed three major hurdles of CAR T cell therapy for GBM. While we will use our data to justify the development of a future clinical study utilizing optimized IL13Rα2-CAR T cells for patients with GBMs; our modified approach to T cell therapy should be applicable to a broad range of solid tumors.