Novel Immunotherapy of Glioma using miR-17-92 Transfected T Cells

描述（由申请人提供）：我们的长期目标是开发有效的脑肿瘤免疫治疗策略。在目前的资助期内，我们的临床前转化癌症免疫学研究和早期疫苗接种试验证明了我们上调1型抗肿瘤T细胞反应的安全性和免疫活性。我们现在建议通过探索一组特定的microrna （miRs）增强这些T细胞免疫治疗潜力的能力来扩展这项工作。我们的miR微阵列分析显示，miR-17-92 microRNAs集群（miR-17-92），已知可促进细胞存活和增殖，是小鼠1型T细胞中与2型细胞相比最显著过表达的miR之一。我们的数据表明，2型倾斜的肿瘤微环境可以诱导miR- 17-92在T细胞中的表达下调，从而降低肿瘤特异性T细胞的持久性和有效性。此外，与野生型小鼠相比，来自miR-17-92转基因（TG）小鼠的T细胞表现出优越的1型表型，IFN-3的产生和vla4的表达增加。肿瘤免疫治疗的主要障碍包括肿瘤抗原（TAs）的免疫原性差和T细胞在荷瘤宿主中的存活时间短。然而，我们和其他人的数据强烈表明，miR-17-92在T细胞中的表达会降低激活阈值，增强T细胞的增殖、存活和1型功能。因此，我们假设工程ta特异性细胞毒性T淋巴细胞（CTL）表达miR-17-92簇将通过增强ta特异性1型CTL功能提供有效和持久的抗肿瘤活性。我们将使用来自小鼠TG的T细胞对miR-17-92和hgp100 25-33 （Pmel）特异性T细胞受体（TCR） （miR-17- 92/Pmel-TG T细胞）来评估我们的假设。具体而言，我们将确定：Aim 1: miR-17-92过表达是否能增强体外1型抗肿瘤T细胞的激活和存活。我们将验证我们的假设，即在ta特异性ctl中，转基因介导的miR-17-92表达将增加对TAs的敏感性、生存率和1型功能，而不会诱导致癌转化。目的2：过表达miR-17-92的ta特异性ctl在体内赋予持久和强大的1型免疫。我们将探讨miR-17-92表达是否改善：1)1型ta特异性ctl的扩增和持久性；2)记忆T细胞的发育；3)非荷瘤小鼠CTL活性的回忆反应。目的3：过继转移表达miR-17-92的T细胞增加抗肿瘤作用。我们将确定miR-17-92/Pmel-TG ctl的ACT是否能在GL261小鼠或新生小鼠胶质瘤中提供持久和有效的抗胶质瘤免疫应答。这些提出的临床前研究将不仅对胶质瘤免疫治疗，而且对整个癌症免疫治疗领域产生重大影响，特别是通过ta特异性T细胞的基因工程。

DESCRIPTION (provided by applicant): Our long term goal is to develop effective immunotherapy strategies for brain tumors. Our preclinical translational cancer immunologic studies and early phase vaccination trials in the current funding period demonstrated the safety and immunological activity of our efforts to up-regulate type-1 antitumor T cell responses. We now propose to extend this work by exploring the ability of a specific set of microRNAs (miRs) to enhance the immunotherapeutic potential of these T cells. Our miR microarray analyses revealed that the miR-17-92 cluster of microRNAs (miR-17-92), which is known to promote cell survival and proliferation, is one of the most significantly over-expressed miR in murine type-1 T cells compared with type-2 cells. Our data suggest that the type-2-skewing tumor microenvironment can induce downregulation of miR- 17-92 expression in T cells, thereby diminishing the persistence and efficacy of tumor-specific T cells. Furthermore, T cells derived from miR-17-92 transgenic (TG) mice demonstrated superior type-1 phenotype with increased IFN-3 production and VLA-4 expression when compared with counterparts derived from wild type mice. Major barriers to cancer immunotherapy include the poor immunogenicity of tumor-antigens (TAs) and the short duration of T cell survival in tumor-bearing hosts. However, our data and those from others strongly suggest that miR-17-92 expression in T cells will lower the threshold for activation and enhance T cell proliferation, survival and type-1 functions. We therefore hypothesize that engineering TA-specific cytotoxic T lymphocytes (CTLs) to express the miR-17-92 cluster will provide potent and durable antitumor activity by potentiating TA-specific type-1 CTL functions. We will evaluate our hypothesis using T cells derived from mice TG for miR-17-92 and T cell receptor (TCR)-specific for hgp100 25-33 (Pmel) (miR-17- 92/Pmel-TG T cells). Specifically, we will determine whether: Aim 1: miR-17-92 overexpression enhances activation and survival of type-1 anti-tumor T cells in vitro We will test our hypothesis that transgene-mediated expression of miR-17-92 in TA-specific CTLs will increase sensitivity to TAs, survival, and type-1 function without inducing oncogenic transformation. Aim 2: TA-specific CTLs overexpressing miR-17-92 confer durable and robust type-1 immunity in vivo We will address whether miR-17-92 expression improves: 1) expansion and persistence of type-1 TA-specific CTLs; 2) development of memory T cells; and 3) recall responses for CTL activity in non-tumor bearing mice. Aim 3: Adoptive transfer of T cells engineered to express miR-17-92 increases anti-tumor effects. We will determine whether ACT of miR-17-92/Pmel-TG CTLs will provide durable and effective anti-glioma immune responses compared with control Pmel-TG CTLs in mice bearing GL261 or de novo murine gliomas. These proposed preclinical studies will have significant impact on not only glioma-immunotherapy, but also the entire field of cancer immunotherapy, particularly through genetic engineering of TA-specific T cells.