TCR/DAP10 chimera as a means to attaining persistent anti-tumor T cell responses

描述(申请人提供)：目前，转移到癌症患者体内后未能持续存在是限制T细胞遗传采用细胞转移有效性的一个因素 经修饰后表达肿瘤反应性T细胞受体。本申请的目的是通过将DAP10的细胞质信号域添加到TCRTIL 1383I(黑色素瘤反应性)的�链和�链的细胞质结构域的末端来修饰TCRTIL 1383I(黑色素瘤反应)。我们认为，TCR/DAP10在TCR参与后将直接激活DAP10通路，这些独特的信号将提高转移的T细胞的存活率。初步数据：我们已经在CD8+T细胞中证明了通过[自然表达的]激活受体复合体NKG2D的信号增强记忆发育。我们的数据显示，TCR转导的CD8+T细胞中的NKG2D信号增强了它们的抗肿瘤效力和体内持久性。这些数据表明，通过CD8+T细胞中的NKG2D信号是克服TCR-TD T细胞有限生存的可行方法。我们将结合TCR连接和激活下游的NKG2D信号。由于NKG2D本身不能发出信号，我们将使用DAP10的信号域，DAP10是在CD8+T细胞中介导NKG2D下游信号的适配分子。我们将通过将DAP10的细胞质信号域添加到�-链和�-链的TCR胞浆域的末端来修饰人TCRTIL1383I(酪氨酸酶反应)。假设：我们假设TCR/DAP10将在TCR参与时直接激活DAP10刺激通路，这些独特的信号将提高过继转移的T细胞的存活率。策略：将含有TCR/DAP10的逆转录病毒载体导入人T细胞。表达TCR/DAP10的人类T细胞将使用人源化的黑色素瘤模型来检测它们的信号通路、生存能力和调解已建立的人类癌症的消退能力。免疫缺陷的A2/NSG小鼠和人黑色素瘤细胞株将用于抗肿瘤实验。我们将通过以下具体目标SA1来验证我们的假设。开发TCR/DAP10结构来诱导和分析DAP10信号。SA2.研究TCR/DAP10利用的信号通路及其在人类T细胞中的细胞后果。SA3.检测表达TCR/DAP10的T细胞抗人黑色素瘤。我们的策略的意义和创新特征是，这些工程T细胞将具有酪氨酸酶的特异性，它们的TCR的结合将激活DAP10共刺激通路以及下游的TCR信号。

DESCRIPTION (provided by applicant): Currently, failure to persist after transfer into cancer patients is a factor that limits the effectiveness of adoptive cell transfer of T cells genetically modified to express a tumor-reactive T cell receptor. The goal of this application is to modify th human TCR TIL 1383I (melanoma-reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the cytoplasmic domains of the �- and �- chains of the TCR. We believe that the TCR/DAP10 will directly activate the DAP10 pathway upon TCR engagement, and that these unique signals will enhance the survival of transferred T cells. Preliminary data: We have demonstrated in CD8+ T cells that signaling through the [naturally expressed] activation receptor complex NKG2D enhances memory development. Our data show that NKG2D signaling in TCR-transduced CD8+ T cells augments their anti-tumor potency and in vivo persistence. These data argue that signaling through NKG2D in CD8+ T cells is a viable approach to overcoming the limited survival of TCR- Td T cells. We will couple TCR ligation and activation of downstream NKG2D signaling. Because NKG2D is unable to signal by itself, we will use the signaling domain of DAP10, the adaptor molecule that mediates signaling downstream from NKG2D in CD8+ T cells. We will modify the human TCR TIL 1383I (tyrosinase- reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the TCR cytoplasmic domains of the �- and �- chains. Hypothesis: We hypothesize that the TCR/DAP10 will directly activate the DAP10 stimulatory pathway upon TCR engagement, and that these unique signals will enhance the survival of adoptively transferred T cells. Strategy: The retroviral vector containing TCR/DAP10 construct will then be transduced into human T cells. Human T cells expressing TCR/DAP10 will be examined for their signaling pathways, capacity to survive and ability to mediate the regression of established human cancer using a humanized model of melanoma. Immune deficient A2/NSG mice and human melanoma cell lines will be used for anti-tumor experiments. We will test our hypothesis through the following specific aims SA1. Develop TCR/DAP10 constructs to induce and dissect DAP10 signaling. SA2. Study the signaling pathways utilized by TCR/DAP10 and their cellular consequences in human T cells. SA3. Test T cells expressing TCR/DAP10 against human melanoma. The significance and innovative character of our strategy would be that these engineered T cells will have specificity for tyrosinase, and engagement of their TCR will activate the DAP10 costimulatory pathway as well as downstream TCR signaling.