TARGETING OF PROTEINS TO GRANULES OF CYTOTOXIC T LYMPHOCYTES

将蛋白质靶向细胞毒性 T 淋巴细胞颗粒

• 批准号: 6102901
• 负责人: ARIE S BELLDEGRUN
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6102901
• 关键词: SCID mouse; biological signal transduction; blood proteins; clone cells; cytokine; defensins; disease /disorder model; drug delivery systems; enzyme linked immunosorbent assay; fusion gene; gene therapy; genetic manipulation; genetic markers; granule; human therapy evaluation; immunoelectron microscopy; immunoprecipitation; lymph nodes; lymphocyte; neoplasm /cancer immunology; neoplasm /cancer immunotherapy; neoplasm /cancer vaccine; neutrophil; posttranslational modifications; reporter genes; tumor infiltrating lymphocyte

We propose to develop a novel method for the delivery of gene-engineered proteins to tumors by targeting the proteins to the granules of tumor infiltrating lymphocytes (TIL). The method is designed to increase tumor selectivity by taking advantage of the granule-release mechanisms that are triggered by specific interactions of lymphocytes with their targets. We have shown in preliminary studies that small transgenic human peptide defensin HNP-1. is targeted to granules of murine granulocytes and cytotoxic T-lymphocytes. Deletion mutagenesis of HNP-1 suggests that a limited segment of this small peptide is essential for granule targeting. In this project we will apply and extend these studies to human TIL with the aim of identifying a peptide sequence, the "targeting motif", that can direct other gene-engineered proteins to granules. We will first study the expression and subcellular distribution of defensin HNP-1 engineered into human TIL. We will then employ deletion mutagenesis and reporter fusion constructs to characterize the defensin targeting motif and its ability to direct reporter proteins (CAT. IL-8) into granules of TIL. Finally, we will examine the effect of the granule environment on the bioactivity of the reporter proteins and compare the selectivity of reporter protein release by granule-targeted and non-targeted TIL in vitro and in vivo, in human tumor-bearing SCID mice. Successful completion of this project will facilitate future clinical trials of tumor-selective delivery by TIL of therapeutic proteins or immunostimulatory cytokines.

我们建议开发一种新的方法来传递基因工程的方法 蛋白质通过将蛋白靶向肿瘤颗粒来靶向肿瘤 浸润淋巴细胞（TIL）。 该方法旨在增加肿瘤 通过利用颗粒释放机制的选择性 淋巴细胞与目标的特定相互作用触发。 我们 在初步研究中已经表明了小的转基因人肽 防御素HNP-1。 针对鼠粒细胞的颗粒和 细胞毒性T淋巴细胞。 HNP-1的缺失诱变表明 该小肽的有限段对于颗粒靶向至关重要。 在这个项目中，我们将把这些研究应用于人类，直到 识别肽序列，即“靶向基序”的目的 将其他基因工程蛋白直接定为颗粒。 我们将首先研究 Defensin HNP-1的表达和亚细胞分布 人类。 然后，我们将使用缺失诱变和记者融合 构造以表征防御素靶向基序及其能力 将报告基因蛋白（CAT。IL-8）直接进入TIL的颗粒。 最后，我们 将检查颗粒环境对生物活性的影响 报告蛋白并比较报告基因蛋白的选择性 通过颗粒靶向和非靶向的til在体外和体内释放 人类肿瘤的SCID小鼠。 成功完成该项目将 促进通过TIL的肿瘤选择性递送的未来临床试验 治疗蛋白或免疫刺激性细胞因子。