Dendritic Cell-Based Genetic Immunotherapy for Melanoma

描述(申请人提供)：这是对ROI CA79976“基于树突状细胞的黑色素瘤遗传免疫疗法”的竞争性续订，我们在其中请求05-08年的支持。我们在上一个资助期的成就包括：1.确定了使用已定义的肿瘤抗原MART-1基因工程的树突状细胞(DC)在小鼠黑色素瘤模型中发生的免疫学事件；2.完成了接受MART-1/27-35多肽冲击的黑色素瘤患者的L/II期临床试验；3.开启了使用腺病毒(Adv)MART-1转导的DC的基因治疗试验。基于这一进展，我们建议继续围绕三个特定目标对人类黑色素瘤的基因免疫治疗进行翻译研究。目的1：CDS缺陷环境中的遗传免疫治疗。我们已经做出了显著和原创性的观察，用AdVMART1转导的DC免疫的CD8或I类敲除小鼠对B16黑色素瘤的保护水平高于野生型(Wt)小鼠。由于CD8细胞耗尽的wt小鼠不能产生保护性免疫，CD8KO小鼠已经发展出一种从产生强大的肿瘤免疫到DC疫苗接种的补偿机制。我们提出的初步证据表明，这种抗肿瘤免疫是通过免疫系统的先天(NK样)和适应性(CD4)臂的效应细胞之间的协作来介导的。我们建议对潜在的机制进行描述。目的：研究AdVMART1/DC免疫黑色素瘤患者的第I类和第II类限制性T细胞反应的生物学特性。在这项临床试验中，患有IV期MART-1阳性黑色素瘤的患者用AdVMART1/DC免疫，这提供了一个独特的机会来确定由针对已定义的“自身”肿瘤抗原的基因免疫所触发的免疫学事件。这种小分子蛋白只有两个表位：人类白细胞抗原A2.1限制的MART-1/27-35和人类白细胞抗原DK4限制的MART-1/51-73。利用ELISPOT和四聚体分析这种I类和II类表位，我们将定量、分离和研究免疫患者中MART-1反应的CD8和CD4T细胞。我们还将研究决定簇传播和交叉呈现在临床反应中的作用，树突状细胞用于疫苗接种的生物学，以及天然免疫在基于树突状细胞的免疫治疗中的可能参与。目的3：CTLA4阻断在临床树突状细胞免疫治疗中的应用。以DC为基础的免疫疗法偶尔会产生戏剧性的临床抗肿瘤反应。我们仔细研究了一名受试者，在接种MART-1/DC疫苗后，接种了CTLA4封闭抗体。免疫学分析表明，CTLA4阻断维持了DC疫苗启动的抗肿瘤免疫应答。为了验证这一假设，我们设计了一项II期随机试验，主要目的是利用ELISPOT分析检测MART-1/27-35/DC+CTLA4阻断对黑色素瘤抗原特异性激活T细胞频率的影响。这项试验将为控制DC免疫治疗活动的自动调节机制提供洞察力。综上所述，我们建议继续我们在遗传免疫治疗方面的翻译计划，重点放在免疫机制和临床假设检验上。

DESCRIPTION (provided by applicant): This is a competitive renewal for ROI CA79976 "Dendritic cell-based genetic immunotherapy for melanoma" in which we request support for years 05-08. Our accomplishments in the previous funding period include: 1. defining the immunological events taking place in a murine melanoma model using dendritic cell (DC) engineered with a defined tumor antigen MART-1, 2. completing a phase l/II clinical trial in melanoma patients receiving MART-1/27-35 peptide pulsed DC and 3. opening a gene therapy trial using adenovirus (AdV) MART-1-transduced DC. Based on this progress, we propose to continue our translational studies of genetic immunotherapy of human melanoma centered around three specific aims. Aim 1: Genetic Immunotherapy in a CDS-Deficient Environment. We have made the remarkable and original observations that CD8 or Class I knock out mice immunized with AdVMART1-transduced DC have superior levels of protection to B16 melanoma than wild type (wt) mice. Since wt mice depleted of CD8 cells are unable to generate protective immunity, CD8 KO mice have developed a compensatory mechanism from generating robust tumor immunity to DC vaccination. We present preliminary evidence that this antitumor immunity is mediated by collaboration between effector cells of the innate (NK-like) and adaptive (CD4) arms of the immune systems. We propose to characterize the underlying mechanism. Aim 2: The Biology of Class I and Class II-Restricted T Cell Responses in AdVMART1/DC Immunized Patients with Melanoma. This clinical trial, in which patients with stage IV MART-1-positive melanoma are immunized with AdVMART1/DC, provides a unique opportunity to define immunological events triggered by genetic immunization to a defined "self" tumor antigen. Only two epitopes have been described for this small protein-HLA-A2.1-restricted MART-1/27-35 and HLA-DK4 restricted MART-1/51-73. Using ELISPOT and tetramer assays for this class I and II epitopes, we will quantitate, isolate and study MART-1-reactive CD8 and CD4 T cell in immunized patients. We will also study the role of determinant spreading and cross-presentation in clinical response, the biology of DC used for vaccination and the possible participation of innate (NK) immunity in DC-based immunotherapy. Aim 3: CTLA4 Blockade in Clinical Dendritic Cell-Based Immunotherapy. DC-based immunotherapy generates occasional but dramatic clinical antitumor responses. We have closely studied one subject in whom the administration of MART-1/DC vaccines was followed by a CTLA4 blocking antibody. Immunological analysis suggests that the antitumor immune response initiated by the DC vaccines was maintained by CTLA4 blockade. To test this hypothesis, we have designed a phase II randomized trial with the primary goal of detecting the effect of MART-1/27-35/DC + CTLA4 blockade on the frequency of melanoma antigen-specific activated T cells using ELISPOT assays. This trial will provide insight in the autoregulatory mechanisms that govern the activity of DC-based immunotherapy. In summary, we propose to continue our translational program in genetic immunotherapy with an emphasis on immune mechanism and clinical hypothesis-testing.