IL13Ra2 targeted T-cell therapy for glioma

 描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是所有原发性脑肿瘤中最常见的，由于肿瘤的弥漫浸润性和血脑屏障（BBB）的存在，治疗选择有限。考虑到T细胞穿透BBB和浸润肿瘤的能力，表达靶向肿瘤特异性抗原的嵌合抗原受体（CAR）的T细胞具有充当特异性靶向定位于脑的原发性肿瘤或转移瘤的独特细胞毒性工具的潜力。IL 13 R α 2是一种高亲和力的IL 13受体，由于其在GBM中以高频率表达，而在正常组织中不表达，因此是GBM靶向治疗的有希望的候选者。大多数先前的IL 13 R α 2靶向方法利用IL 13突变，其也识别IL 13 R α 1阳性靶细胞，引起了对"靶向/脱癌"毒性的担忧。因此，我们开发了一种不与IL 13 R α 1交叉反应的高亲和力IL 13 R α 2特异性单克隆抗体（MAb）。重要的是，我们的初步结果表明，表达含有IL13R α 2特异性MAb的单链可变片段（scFv）的CAR的T细胞（IL13R α 2-CAR T细胞）对IL13R α 2具有特异性，并且不识别或杀死表达IL13R α 1的靶细胞。在该提案中，我们现在假设IL13R α 2-CAR T细胞在临床前GBM模型中具有有效的抗胶质瘤活性。CAR T细胞主要在缺乏GBM显著特征的异种移植模型中进行评估;最重要的是，它们不能重现其免疫抑制肿瘤微环境，限制了CAR T细胞的临床前测试和未来开发。我们假设这些限制可以通过调整免疫活性GBM模型来克服，该模型非常模拟用于IL13 R α 2-CAR T细胞体内测试的人类疾病。我们的假设将在两个相互关联的目标进行评估。在目的1中，我们将构建一组IL 13 R α 2-CAR，其含有我们的新scFvIL 13 R α 2作为胞外域、短或长铰链、跨膜结构域和胞内结构域，所述胞内结构域含有CD3 β信号传导结构域和源自共刺激分子的信号传导结构域（CD28.CD134.CD14.CD28.CD137.CD28.CD137.CD28.CD137.CD134.CD13.CD28.CD134.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13.CD13）。将使用标准免疫学测定在体外评估表达这些CAR的鼠T细胞的效应器功能。虽然我们的体外实验结果将使我们能够回答与我们的实验系统的最佳功能相关的问题，但这些数据将不能完全代表体内环境。为了弥补这一差距，我们将在Aim 2中使用GL261和SMA-560免疫活性胶质瘤模型在体内比较优化的IL13 R α 2-CAR。我们希望在资助结束时，我们将确定IL13R α 2-CAR的最佳设计，并完成IL13R α 2-CAR T细胞在相关免疫活性胶质瘤模型中的测试。虽然我们使用这些数据来证明I期临床试验的发展，但开发的模型也将有助于未来的研究，我们的目标是将联合收割机IL 13 R α 2-CAR T细胞与其他神经胶质瘤靶向治疗相结合。

 DESCRIPTION (provided by applicant): Glioblastoma multiform (GBM) is the most common of all primary brain tumors with limited therapeutic options due to the diffusely infiltrative nature f the tumor and the presence of the blood brain barrier (BBB). Given an ability of T cells to penetrate the BBB and infiltrate tumors, T cells expressing chimeric antigen receptors (CARs) targeting tumor specific antigens have the potential to serve as a unique cytotoxic tool specifically targeting primary tumors or metastases that localize to the brain. IL13Rα2, the high affinity IL13 receptor is a promising candidate for GBM-targeted therapy since it is expressed at a high frequency in GBM, but not in normal tissues. The majority of prior IL13Rα2-targeted approaches take advantage of IL13 mutinies, which also recognize IL13Rα1-positive target cells, raising concerns of 'on target/off cancer' toxicities. We have therefore developed a high affinity IL13Rα2-specific monoclonal antibody (MAb) that does not cross-react with IL13Rα1. Importantly, our preliminary results indicate that T cells expressing CARs that contain a single chain variable fragment (scFv) of IL13Rα2-specific MAb (IL13Rα2-CAR T cells), are specific for IL13Rα2 and do not recognize or kill target cells expressing IL13Rα1. In this proposal we now hypothesize that IL13Rα2-CAR T cells will have potent anti-glioma activity in preclinical GBM models. CAR T cells have been primarily evaluated in xenograft models that lack salient features of GBMs; most importantly, they do not recapitulate their immunosuppressive tumor microenvironment, limiting preclinical testing and future development of CAR T cells. We hypothesize that these limitations can be overcome by adapting immune competent GBM models, which closely mimics human disease for in vivo testing of IL13Rα2-CAR T cells. Our hypotheses will be evaluated in two interrelated Aims. In Aim 1 we will construct a panel of IL13Rα2-CARs that contain our new scFvIL13Rα2 as an ectodomain, a short or long hinge, a transmembrane domain, and endodomains that contain a CD3ζ signaling domain, and signaling domains derived from co-stimulatory molecules (CD28.ζ, CD28.CD134.ζ, or CD28.CD137. ζ). The effector function of murine T cells expressing these CARs will be evaluated in vitro using standard immunological assays. While the results of our in vitro experiments will allow us to answer questions pertinent to optimal functioning of our experimental system, these data will not be fully representing the in vivo setting. To bridge this gap, we will compare in Aim 2 optimized IL13Rα2-CARs in vivo using the GL261 and SMA-560 immune competent glioma models. We expect at the conclusion of the grant that we will have determined the optimal design of IL13Rα2-CARs and have completed the testing of IL13Rα2-CAR T cells in relevant, immune competent glioma models. While we use this data to justify the development of a Phase I clinical trial, the developed model will also be useful for future studies in which we aim to combine IL13Rα2-CAR T cells with other glioma-targeted therapies.