Dendritic Cell-Based Genetic Immunotherapy for Melanoma

描述（由申请人提供）：这是ROI CA 79976“基于树突状细胞的黑色素瘤遗传免疫疗法”的竞争性更新，我们请求在05-08年提供支持。我们在上一个资助期的成就包括：1。使用用确定的肿瘤抗原MART-1工程化的树突状细胞（DC），确定在鼠黑素瘤模型中发生的免疫学事件，2.在接受MART-1/27-35肽脉冲DC的黑素瘤患者中完成I/II期临床试验，和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具体目标进行人类黑色素瘤基因免疫治疗的转化研究。目的1：在CDS-deficient环境中的遗传免疫治疗。我们已经做出了显著的和原始的观察结果，即用AdVMART 1转导的DC免疫的CD 8或I类敲除小鼠比野生型（wt）小鼠对B16黑素瘤具有上级保护水平。由于耗尽CD 8细胞的野生型小鼠不能产生保护性免疫，因此CD 8 KO小鼠已经发展出从产生对DC疫苗接种的强肿瘤免疫的补偿机制。我们目前的初步证据表明，这种抗肿瘤免疫是由先天（NK样）和适应性（CD 4）的免疫系统的武器的效应细胞之间的合作介导的。我们提出的基本机制的特点。目的2：AdVMART 1/DC免疫黑色素瘤患者中I类和II类限制性T细胞应答的生物学。该临床试验中，IV期MART-1阳性黑色素瘤患者用AdVMART 1/DC免疫，提供了一个独特的机会来定义由定义的“自身”肿瘤抗原的遗传免疫触发的免疫事件。对于这种小蛋白，仅描述了两个表位-HLA-A2. 1限制性MART-1/27-35和HLA-DK 4限制性MART-1/51-73。使用ELISPOT和四聚体测定这种I类和II类表位，我们将定量，分离和研究免疫患者中的MART-1反应性CD 8和CD 4 T细胞。我们还将研究决定簇传播和交叉呈递在临床反应中的作用，用于疫苗接种的DC的生物学以及先天性（NK）免疫在基于DC的免疫治疗中的可能参与。目的3：CTLA 4阻断在临床树突状细胞免疫治疗中的应用基于DC的免疫疗法产生偶尔但显著的临床抗肿瘤反应。我们仔细研究了一名受试者，其中MART-1/DC疫苗的施用随后是CTLA 4阻断抗体。免疫学分析表明，由DC疫苗引发的抗肿瘤免疫应答通过CTLA 4阻断来维持。为了验证这一假设，我们设计了一项II期随机试验，主要目的是使用ELISPOT测定法检测MART-1/27-35/DC + CTLA 4阻断对黑素瘤抗原特异性活化T细胞频率的影响。这项试验将提供关于控制基于DC的免疫疗法活性的自动调节机制的见解。总之，我们建议继续我们在遗传免疫治疗的翻译计划，重点是免疫机制和临床假设检验。

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.