Enzymatic modification of anti-DEC205 to manipulate its immunogenic properties

描述（由申请人提供）：抗DEC 205抗体（？DEC 205）识别树突状细胞（DC）上的表面蛋白，可用于将抗原靶向DC：DEC 205，无论是通过化学缀合还是作为基因融合，都允许其递送至DC。这产生了一个强有力的适应性免疫反应，包括CD 4和CD 8 T细胞的抗原特异性附着？12月205日。这种方法-以及针对树突状细胞上其他表面蛋白（如II类MHC产物）的其他抗体的类似修饰-有望成为一种可能的疫苗策略。大多数化学结合方法缺乏精确性，以及将感兴趣的有效载荷附着到所需的劳动密集型基因融合方法？DEC 205和纯化所得加合物的方法提出了消除这些缺点的替代方案。一个高效和简单的化学酶的替代，使用分选酶从金黄色葡萄球菌，提出了允许一个详细的分析机制，所需的免疫原性？DEC 205加合物。将与Vhh 7进行比较，Vhh 7是一种来源于羊驼的单结构域抗小鼠II类MHC抗体。Vhh 7工程以类似的方式作为分选酶底物，通过产生荧光和生物素化衍生物作为证明其特异性和紧密结合I类MHC产物的手段进行验证。DEC 205或Vhh 7上允许以化学计量方式位点特异性连接T细胞表位、所选的荧光或生物素化有效载荷以及甚至蛋白质大小的取代基。这个程序，被称为sortaging，应允许任何T细胞表位或可追踪的有效载荷（包括蛋白质大小的附件）的具体交付DEC 205+和II类MHC+抗原呈递细胞在体内，而不需要许多独立的遗传？DEC 205或Vhh 7融合构建体。所提出的方法具有额外的优势，非天然的取代基，如容易裂解的接头或佐剂，可以安装，以提高这种加合物的效力，甚至C-末端到C-末端融合是可能的-使用点击化学-比较效力的介绍C-N与C-C融合？DEC 205或Vhh 7抗原。这些目标将在两个具体目标中解决，其将应用于从通过体细胞核转移产生的克隆小鼠获得的对MHV 68 ORF 8抗原特异性的CD 8 T细胞：1.建立基础机制？抗原交叉呈递的DEC 205和Vhh 7依赖性途径。2.探索的能力？DEC 205和VHH 7加合物以激活效应CD 8 T细胞（或从头引发它们）并建立针对MHV 68病毒的保护。如果成功，这些策略将打开大门，创造理想的T细胞反应，而不使用感染剂，并普遍改善免疫操作的选择，以实现治疗效果。!

DESCRIPTION (provided by applicant): The anti DEC205 antibody (?DEC205) recognizes a surface protein on dendritic cells (DCs) that can be used to target antigens to DCs: attachment of an antigenic moiety to ?DEC205, either through chemical conjugation or as a genetic fusion, allows its delivery to the DC. This generates a potent adaptive immune response that includes CD4 and CD8 T cells specific for the antigen attached to?DEC205. This approach -as well as similar modifications of other antibodies directed against yet other surface proteins on dendritic cells, such as Class II MHC products- holds promise as a possible vaccine strategy. The lack of precision of most chemical conjugation methods, and the labor-intensive genetic fusion approaches required to attach the payload of interest to ?DEC205 and purify the resulting adduct suggest an alternative to eliminate these shortcomings. A highly efficient and straightforward chemo-enzymatic alternative, using sortase from Staphylococcus aureus, is proposed to allow a detailed analysis of the mechanisms that underlie the desirable immunogenic properties of ?DEC205 adducts. A comparison will be made with Vhh7, a single domain anti- mouse Class II MHC antibody derived from an alpaca. Vhh7 was engineered in similar fashion to serve as a sortase substrate, validated through generation of fluorescent and biotinylated derivatives as the means of demonstrating its specificity and tight binding to Class I MHC products The introduction of a LPXTG motif at the C-terminus of the heavy chain of ?DEC205 or onto Vhh7 allows site-specific attachment of a T cell epitope, a fluorescent or biotinylated payload of choice and even protein-sized substituents in a stoichiometric manner. This procedure, referred to as sortagging, should permit the specific delivery of any T cell epitope or traceable payload (including protein-sized attachments) to DEC205+ and Class II MHC+ antigen presenting cells in vivo, without the need for numerous independent genetic ?DEC205 or Vhh7 fusion constructs. The proposed method has the added advantage that non-natural substituents, such as easily cleavable linkers or adjuvants, can be installed to improve potency of such adducts, and that even C-terminus to C-terminus fusions are possible -using click chemistry- to compare efficacy of presentation of C-N versus C-C fusions of ?DEC205 or Vhh7 with antigen. These goals will be addressed in two specific aims, to be applied to CD8 T cells, specific for the MHV68 ORF8 antigen, obtained from cloned mice created by somatic cell nuclear transfer: 1. Establish the mechanism that underlies ?DEC205- and Vhh7 dependent routes of antigen cross- presentation. 2. Explore the ability of ?DEC205 and VHH7 adducts to activate effector CD8 T cells (or elicit them de novo) and establish protection against MHV68 virus. If successful these strategies would open the door to creating desirable T cell responses without the use of infectious agents and generally improve options for immune manipulations to achieve therapeutic effect. !