Use of Adjuvants to Improve Skin Genetic Immunizations

描述(申请人提供)：黑色素瘤是最具侵袭性的肿瘤之一，发病率和死亡率都在增加。虽然黑色素瘤是免疫原性肿瘤，可以通过免疫介导的自发消退来潜在地消除，但这种现象非常罕见。更常见的情况是，由于缺乏肿瘤特异性抗原(AGS)或形成肿瘤特异性耐受状态，免疫系统无法识别黑色素瘤。针对黑色素瘤的DNA疫苗免疫策略是一种很有前途的治疗手段，其基础是诱导肿瘤特异性的CD4T辅助1(Th1)淋巴细胞和CD8细胞毒性T细胞(CTL)。由于皮肤的易达性和固有的免疫系统，皮肤是基因免疫的最佳部位，而基因枪(GO)是诱导转基因AGS在皮肤中高表达的极佳方法。皮肤的专业抗原加工和提呈细胞[皮肤树突状细胞(DCs)：表皮朗格汉斯细胞和真皮DCs]可以通过GG免疫原位转移，或者它们也可以内化邻近皮肤细胞的转基因蛋白。因此，通过GG直接间接导入皮肤树突状细胞，在体内构建皮肤免疫系统是可能的。在外周组织(即皮肤)DC激活的早期阶段，适当的免疫刺激信号的存在“指示”DC完全成熟，并诱导有效的抗肿瘤Th1偏向免疫反应。然而，尽管GG能诱导抗肿瘤的CD8CTL，但GG是否能产生有效的以CD4Th1为主的免疫应答(维持有效的细胞抗肿瘤免疫所必需的)以及它是否能诱导Th2细胞仍存在争议。这一建议的假设是，利用GG在Th1驱动佐剂P物质神经激肽1受体激动剂(SP-NK1a)和咪喹莫特(在GG免疫部位成熟并使皮肤DC Th1极化)的存在下，在皮肤中表达转基因黑色素瘤AGS，将产生完全激活的皮肤DC，具有高效刺激肿瘤抗原特异性T细胞的能力。在这些条件下产生的皮肤来源的DC将偏向于将原始的肿瘤特异性T细胞分化为Th1细胞，并促进CTL的发展，产生有效且持久的抗肿瘤免疫反应。为了验证这一假设，我们提出了以下具体目标：目的1：在Th1驱动佐剂咪喹莫特和SP-NK1a的存在下，研究GG转基因皮肤中DC迁移的功能。目的2：在体内分析Th1驱动佐剂在皮肤GG基因免疫诱导的免疫应答中的作用；目的3：分析Th1驱动佐剂对人皮肤GG基因免疫后不同亚群迁移树突状细胞的分化能力。

DESCRIPTION (provided by applicant): Melanomas are one the most aggressive tumors with increasing incidence and mortality. Although melanomas are immunogenic tumors that could be potentially eliminated by immune-mediated spontaneous regression, this phenomenon is extremely rare. More frequently, melanomas are not recognized by the immune system due to the absence of tumor-specific antigens (Ags) or development of a state of tumor-specific tolerance. Immunization strategies against melanoma by means of DNA vaccines are promising therapeutic tools based on the induction of tumor-specific CD4+ T helper 1 (Th1) lymphocytes and CD8+ cytotoxic T cells (CTLs). Due to its easy accessibility and intrinsic immune system, the skin is an optimal site for genetic immunization and the gene gun (GO is an excellent approach to induce high expression of transgenic Ags in the skin. Professional Ag processing and presenting cells of the skin [cutaneous dendritic cells (DCs): epidermal Langerhans cells and dermal DCs] can be transfected in situ by GG-immunization, or alternatively, they may internalize transgenic proteins from neighboring skin cells. Therefore, through direct of indirect transfection of skin DCs by the GG, it is possible to engineer in vivo the skin immune system. The presence of appropriate immuno-stimulatory signals during the early stages of DCs activation in peripheral tissues (i.e. skin) "instructs" DCs to fully mature and to induce an effective anti-tumor Th1-biased immune response. However, although the GG induces anti-tumor CD8+ CTLs, it is still controversial whether the GG is able to generate an effective CD4+ Th1-biased immune response (required to maintain efficient cellular anti-tumor immunity) and it rather induces Th2 cells. The hypothesis of this proposal is that the use of the GG to express transgenic melanoma Ags in the skin in the presence of the Th1-driving adjuvants substance P neurokinin 1 receptor agonist (SP-NK1a) and Imiquimod (to mature and to Th1-polarize skin DCs at the site of GG-immunization) will generate fully activated skin DCs with ability to stimulate efficiently tumor Ag-specific T cells. Skin-derived DCs generated under these conditions will bias the differentiation of naive tumor-specific T cells into Th1 cells and promote development of CTLs, generating an effective and long-lasting anti-tumor immune response. To test the hypothesis, we propose the following specific aims: Aim 1: To investigate the function of DCs migrated from skin transfected with GG in the presence of Th1-driving adjuvants Imiquimod and SP-NK1a. Aim 2: To analyze in vivo the role of Th1-driving adjuvants in the outcome of the immune responses induced by GG genetic immunization of skin, and Aim 3: To analyze the capacity of the Th1-driving adjuvants to polarize different subpopulations of skin migratory DCs from human skin transfected with the GG.