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

摘要 癌症的免疫治疗方法可能导致晚期转移性肿瘤的偶尔长期缓解。 对常规治疗方式有抵抗力的癌症。制定更有效的办法需要 这些抗肿瘤反应的免疫生物学的详细分析。然而，现有的技术 免疫应答的研究已经证明具有有限的能力来充分表征这种复杂的过程， 这主要是因为协调这些反应的细胞，肿瘤抗原特异性CD8+细胞， 细胞毒性T淋巴细胞（CTL）是外周循环中的稀有细胞。我们建议开发一个模块化的 设计、生产和验证功能组件的技术， 肿瘤特异性CD8+ CTL使用有限的样品，最终允许定量和功能 表征肿瘤浸润性CD8+ CTL。在目标1中，我们提出使用负载有以下的捕获MHC四聚体： 肿瘤抗原（CD8+ CTL的特异性配体）用于开发小鼠和人微免疫芯片（uIC） 具有数字控制的、可单独访问的微流体腔室的模块，以捕获、分类、定量和分析微流体。 孵育肿瘤特异性T细胞。在目标2中，我们建议开发用于量化表面的单个模块， 细胞内和分泌的蛋白质，来自非常少量的抗肿瘤特异性T细胞（甚至单个细胞）， 以及一个用于定量来自少量细胞的mRNA特征的模块（与Fluidigm合作）。在 目标3不同的模块将被整合和测试，首先使用肿瘤特异性的 CD8+ CTL，然后是从免疫治疗的小鼠模型获得的复杂性增加的样品，和 最终从参与癌症临床试验的患者获得人肿瘤特异性CD8+ CTL 免疫疗法在加州大学洛杉矶分校进行

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.