Translational linkage strategies for DNA vaccines against cancer

描述(申请人提供)：该项目的目标是开发有效的分子定义的免疫佐剂，用于主动免疫抗癌，并与更有效的免疫佐剂直接相关，用于免疫感染病原体。虽然对癌症的耐受性是可逆的，但针对癌症自身抗原的免疫在耐受性和免疫调节方面面临着巨大的障碍。有效的免疫佐剂对于针对癌症自身抗原的成功主动免疫是必要的，因为癌症自身抗原的免疫原性天生就很差。最近，我们获得的数据表明，在基因融合佐剂免疫的动物中，适应性免疫反应中的细胞被激活得很厉害。我们使用遗传佐剂(质粒DNA)，将包括VP22(疱疹病毒)和外毒素A(铜绿假单胞菌)在内的微生物基因与优化的自身抗原(酪氨酸酶相关蛋白1=TYRP1)相结合，生成融合基因产品，增强协同和高效的免疫反应。编码各种候选微生物基因融合的DNA是基于广泛的初步筛选和我们对这些分子如何调节获得性免疫细胞激活的理解而精心挑选的。能够最有效地激活免疫细胞的微生物基因融合佐剂将被结合到一个由口蹄疫病毒(FMDV)的18个氨基酸“2A序列”连接的单一质粒上，以产生一种双顺反子DNA疫苗。具体目标1检验与单基因融合相比，结合两个或更多由2A序列连接的基因融合是否可以提高免疫细胞的激活水平。特定目的2研究最有效的与2A连锁的基因融合嵌合体中T细胞的激活机制。特异靶3评估多拷贝DNA微生物融合疫苗是否有效，作为可以与IL-12/Fc融合DNA构建一起应用于不同肿瘤的组合剂。

DESCRIPTION (provided by applicant): The goal of this project is to develop effective molecularly-defined immune adjuvants for active immunization against cancer, and with direct relevance for more potent immune adjuvants for immunization against infectious pathogens. Although tolerance to cancer is reversible, immunization against cancer self-antigens faces substantial hurdles related to tolerance and immune regulation. Potent immune adjuvants will be necessary for successful active immunization against cancer self-antigens, which are inherently poorly immunogenic. Recently, we have acquired data that demonstrates robust activation of cells from the adaptive immune response in animals immunized with gene-fusion adjuvants. We use genetic adjuvants (plasmid DNA) that combine microbial genes, including VP22 (Herpesvirus) and Exotoxin A (Pseudomonas aeruginosa), with an optimized self-antigen (tyrosinase-related protein 1 = TYRP1) to generate fusion gene products that potentiate a synergistic and highly effectual immune response. DNA encoding a variety of candidate microbial gene-fusions have been carefully selected based on extensive preliminary screening and on our understanding of how these molecules regulate the activation of acquired immune cells. The microbial gene-fusion adjuvants that elicit the most potent activation of immune cells will be combined onto a single plasmid, linked by an 18 amino acid "2A sequence" from the foot-and-mouth disease virus (FMDV), to generate a bicistronic DNA vaccine. Specific Aim 1 examines whether combining two or more gene-fusions coupled by the 2A sequence can increase the level of immune cell activation compared to a single gene-fusion. Specific Aim 2 studies the mechanism of T-cell activation from the most potent 2A-linked gene-fusion chimeras. Specific Aim 3 evaluates whether multi-copy DNA microbial fusion vaccines are effective, as combinatorial agents that can be applied with an IL-12/Fc fusion DNA construct against different tumors.