ANALYSIS OF E7 SPECIFIC T CELLS USING MHC-1GG MOLECULES

本申请寻求评价一种新颖的 涉及将HPV-16 E7抗原靶向至MHC II类的疫苗方法 加工路径在评估中最重要的努力之一 治疗性疫苗是对诱导的体内免疫应答的测量 与抗肿瘤功效相关。最近的研究提出了质疑， 标准品原液和有限稀释CTL分析在估计 抗原特异性T细胞的真实频率和功能状态。一个 一种令人兴奋的直接观察抗原特异性T细胞的新方法， 稳定且特异性结合的多聚体MHC+肽复合物的用途 抗原特异性T细胞。申请人开发了一种简单而通用的 通过将MHC分子遗传地连接到 二聚IgG支架。这些肽-MHC-IgG嵌合体已被证明可以结合 稳定且特异性地与抗原特异性CD 8+和CD 4 + T细胞结合。他提议 利用这些试剂来评价体内动力学和功能 接受靶向E7的LAMP的患者中E7特异性T细胞的状态 疫苗。具体而言，他建议：1）开发稳定的CD 8 + T细胞系， 特异于免疫显性HLA-A2限制性E7肽的克隆。2)发展 并检测E7-HLA-A2-IgG二聚体中三种鉴定的免疫显性HLA-A2 限制性E7肽。3)评价E7+A2特异性CD 8 + T细胞的体内动力学 使用E7-HLA-A2-IgG的接种HLA-A2+患者中的T细胞应答 在特定目标2中开发的嵌合体。4)开发类似E7-MHC II-IgG 能够检测对所呈递的E7肽具有特异性的CD 4 + T细胞的嵌合体 常见的HLA II类等位基因DR 1和DR 4。最终，这些分析将 检验以下假设：表达HPV 16 E7+ SIL或 使用LAMP靶向E7疫苗的宫颈癌增加了 活化外周血和疾病部位的E7特异性T细胞。

DESCRIPTION: (Applicant's Abstract) This application seeks to evaluate a novel vaccine approach involving targeting the HPV-16 E7 antigen to the MHC class II processing pathway. One of the most important endeavors in evaluating therapeutic vaccines is the measurement of induced in vivo immune responses relevant to anti-tumor efficacy. Recent studies call into question the sensitivity of standard bulk and limiting dilution CTL analyses in estimating the true frequency and functional status of antigen specific T cells. An exciting new approach to directly visualize antigen specific T cells has been the use of multimeric MHC+ peptide complexes that bind stably and specifically to antigen specific T cells. The applicant has developed a simple and versatile approach to MHC-peptide multimerization by linking MHC molecules genetically to a dimeric IgG scaffold. These peptide-MHC-IgG chimeras have been shown to bind stably and specifically to antigen-specific CD8+ and CD4+ T cells. He proposes to utilize these reagents to evaluate the in vivo dynamics and functional status of E7 specific T cells in patients receiving the LAMP targeted E7 vaccines. Specifically, he proposes to: 1) Develop stable CD8+ T cell lines and clones specific for immunodominant HLA-A2 restricted E7 peptides. 2) Develop and test E7-HLA-A2-IgG dimers for three identified immunodominant HLA-A2 restricted E7 peptides. 3) Evaluate the in vivo dynamics of E7+A2 specific CD8+ T cell responses in vaccinated HLA-A2+ patients utilizing the E7-HLA-A2-IgG chimeras developed in Specific Aim 2. 4) Develop analogous E7-MHC II-IgG chimeras capable of detecting CD4+ T cells specific for E7 peptides presented by the common HLA Class II alleles DR1 and DR4. Ultimately, these analyses will test the hypothesis that vaccination of patients expressing HPV 16 E7+ SIL or cervical cancer with LAMP-targeted E7 vaccines increases the numbers of activated E7 specific T cells in peripheral blood and at the site of disease.