Aurisicchio Luigi, Fridman Arthur, Bagchi Ansuman, Scarselli Elisa, La Monica Nicola, Ciliberto Gennaro
Takis; Rome, Italy ; BIOGEM, scarl; Ariano Irpino (Av), Italy.
Merck & Co., Inc.; West Point, PA USA.
Oncoimmunology. 2014 Jan 1;3(1):e27529. doi: 10.4161/onci.27529. Epub 2014 Jan 16.
Genetic vaccines are emerging as a powerful modality to induce T-cell responses to target tumor associated antigens (TAA). Viral or plasmid DNA or RNA vectors harbor an expression cassette encoding the antigen of choice delivered in vivo by vaccination. In this context, immunizations with minigenes containing selected, highly antigenic, T-cell epitopes of TAAs may have several advantages relative to full-length proteins. The objective of this study was to identify an optimal scaffold for minigene construction. We generated a number of minigenes containing epitopes from the carcinoembryonic antigen (CEA) model TAA and utilized muscle DNA electro-gene-transfer (DNA-EGT) to vaccinate HLA-A*0201 (HHD) and CEA/HHD double transgenic mice. The components utilized to construct the minigenes included CD8 T cell epitopes and (or) anchor modified analogs that were selected on the basis of their predicted binding to HLA-*A0201, their uniqueness in the human proteome, and the likelihood of cancer cell natural processing and presentation via MHC-I. Other candidate components comparatively tested included: helper CD4 T-cell epitopes, flanking regions for optimal epitope processing (including both proteasome-dependent and furin-dependent polypeptide processing mechanisms), and immunoenhancing moieties. Through a series of comparative studies and iterations we have identified an optimal minigene scaffold comprising the following elements: human tissue plasminogen activator (TPA) signal peptide, T-cell epitopes connected by furin sensitive linkers, and the enterotoxin B subunit. The selected epitope modified minigenes (EMM) delivered by DNA-EGT were able to break immune tolerance in CEA/HHD mice and induce a strong immune response against all epitopes tested, independently of their relative positions within the scaffold. Furthermore, the optimized EMMs delivered via DNA-EGT were more immunogenic and exerted more powerful antitumor effects in a B16-CEA/HHD metastatic melanoma model than a DNA vector encoding the full-length protein or a mixture of the same peptides injected subcutaneously. Our data may shed light on the optimal design of a universal vehicle for epitope-targeted, genetic cancer vaccines.
基因疫苗正成为一种强大的方式，可诱导针对靶肿瘤相关抗原（TAA）的T细胞反应。病毒或质粒DNA或RNA载体携带一个表达盒，该表达盒编码通过疫苗接种在体内递送的所选抗原。在这种情况下，用含有TAA的选定、高抗原性T细胞表位的微型基因进行免疫接种相对于全长蛋白可能具有几个优势。本研究的目的是确定微型基因构建的最佳支架。我们生成了一些含有癌胚抗原（CEA）模型TAA表位的微型基因，并利用肌肉DNA电基因转移（DNA-EGT）对HLA-A*0201（HHD）和CEA/HHD双转基因小鼠进行疫苗接种。用于构建微型基因的组件包括CD8 T细胞表位和（或）基于其与HLA-*A0201的预测结合、在人类蛋白质组中的独特性以及癌细胞通过MHC-I进行天然加工和呈递的可能性而选择的锚定修饰类似物。其他经过比较测试的候选组件包括：辅助性CD4 T细胞表位、用于最佳表位加工的侧翼区域（包括蛋白酶体依赖性和弗林蛋白酶依赖性多肽加工机制）以及免疫增强部分。通过一系列比较研究和迭代，我们确定了一种最佳微型基因支架，其包含以下元件：人组织纤溶酶原激活剂（TPA）信号肽、通过弗林蛋白酶敏感接头连接的T细胞表位以及肠毒素B亚基。通过DNA-EGT递送的所选表位修饰微型基因（EMM）能够打破CEA/HHD小鼠的免疫耐受，并诱导针对所有测试表位的强烈免疫反应，而与它们在支架内的相对位置无关。此外，在B16-CEA/HHD转移性黑色素瘤模型中，通过DNA-EGT递送的优化EMM比编码全长蛋白的DNA载体或皮下注射相同肽的混合物具有更强的免疫原性和更强大的抗肿瘤作用。我们的数据可能为针对表位的基因癌症疫苗的通用载体的最佳设计提供启示。
