MICRO/NANO-IMMUNOCHIP (UIC) DEVELOPMENT AND TESTING IN MICE AND HUMANS

摘要 癌症的免疫治疗方法可以偶尔导致晚期转移瘤的长期缓解 对传统治疗模式具有抵抗力的癌症。开发更有效的方法需要 详细分析这些抗肿瘤反应的免疫生物学。然而，现有的技术用于 对免疫反应的研究已经证明，完全描述这一复杂过程的能力有限， 很大程度上是由于协调这些反应的细胞，肿瘤抗原特异性CD8+ 细胞毒性T淋巴细胞(CTL)是外周循环中罕见的细胞。我们建议开发一个模块化的 允许设计、生产和验证用于列举的功能部件的技术 使用限制样本的肿瘤特异性CD8+CTL，最终允许定量和功能 肿瘤浸润性CD8+CTL的特征。在目标1中，我们建议使用捕获MHC四聚体来装载 肿瘤抗原(CD8+CTL的特异性配体)研制鼠和人微免疫芯片(UIC) 具有数字控制的、可单独访问的微流体室的模块，用于捕获、分类、定量和 培养肿瘤特异性T细胞。在目标2中，我们建议开发用于量化表面的单独模块， 细胞内和分泌的蛋白质，来自极少量的抗肿瘤特异性T细胞(甚至是单个细胞)， 以及一个模块，用于量化来自少量细胞的mRNA签名(与Fluidigm合作)。在……里面 目标3将集成和测试不同的模块，首先使用特定于肿瘤的特定人群 CD8+CTL，然后是从小鼠免疫治疗模型中获得的日益复杂的样本，以及 最终从参与癌症临床试验的患者那里获得人类肿瘤特异性CD8+CTL 加州大学洛杉矶分校正在进行免疫治疗。

Abstract Immunotherapy approaches for cancer can result in occasional long term remissions of advanced metastatic cancers resistant to conventional modes of therapy. Development of more effective approaches requires a detailed analysis of the immunobiology of these antitumor responses. However, existing technologies for the study of immune responses have proven to have limited ability to fully characterize this complex process, manily due to the fact that the cells that orchestrate these responses, the tumor antigen-specific CD8+ cytotoxic T lymphocytes (CTL) are rare cells in peripheral circulation. We propose to develop a modular technology to allow for the design, production and validation of functional components for the enumeration of tumor-specific CD8+ CTL using limiting samples, eventually allowing to quantitate and functionally characterize tumor-infiltrating CD8+ CTL. In Aim 1 we propose to use capture MHC tetramers loaded with tumor antigens (the specific ligands for CD8+ CTL) to develop murine and human micro-lmmunochip (uIC) modules with digitally controlled, individually accessible microfluidic chambers to capture, sort, quantitate and incubate tumor-specific T-cells. In Aim 2 we propose to develop individual modules for quantifying surface, intracellular and secreted proteins, from very small numbers of anti-tumor specific T cells (even single cells), and a module for quantifying mRNA signatures from small numbers of cells (in collaboration with Fluidigm). In Aim 3 the different modules will be integrated and tested, first using defined populations of tumor-specific CD8+ CTL, followed by samples of increasing complexity obtained from mouse models of immunotherapy, and eventually human tumor-specific CD8+ CTL obtained from patients participating in clinical trials of cancer immunotherapy being conducted at UCLA.