Enzymatic modification of anti-DEC205 to manipulate its immunogenic properties

描述（由申请人提供）：抗DEC205抗体(？DEC205)识别树突状细胞（dc）上的一种表面蛋白，该蛋白可用于靶向dc上的抗原：抗原片段附着在？无论是通过化学偶联还是作为基因融合，DEC205都允许其传递到DC。这就产生了一种强有力的适应性免疫反应，包括CD4和CD8 T细胞，它们对附着在？DEC205上的抗原具有特异性。这种方法——以及针对树突状细胞表面蛋白（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。大多数化学偶联方法缺乏精度，并且需要劳动密集型的遗传融合方法将感兴趣的有效载荷附加到？DEC205和纯化所得加合物为消除这些缺点提供了一种替代方法。提出了一种高效、直接的化学酶替代方案，使用来自金黄色葡萄球菌的分选酶，以详细分析？DEC205加合物。将与Vhh7进行比较，Vhh7是一种从羊驼中提取的单域抗小鼠ⅱ类MHC抗体。以类似的方式设计Vhh7作为分选酶底物，通过生成荧光和生物素化衍生物来验证其特异性和与I类MHC产品的紧密结合。DEC205或Vhh7允许位点特异性附着T细胞表位，荧光或生物素化负载选择，甚至蛋白质大小的取代基以化学计量的方式。这一过程被称为分选标记，应该允许将任何T细胞表位或可追踪的有效载荷（包括蛋白质大小的附着物）在体内特异性递送到DEC205+和II类MHC+抗原呈递细胞，而不需要许多独立的遗传？DEC205或Vhh7融合构建体。所提出的方法还有一个额外的优点，即可以安装非天然取代基，如易于切割的连接剂或佐剂，以提高此类加合物的效力，并且甚至可以使用点击化学来比较C-N与C-C融合的功效。DEC205或Vhh7抗原。这些目标将在两个特定的目标中得到解决，这些目标将应用于从体细胞核移植产生的克隆小鼠中获得的CD8 T细胞，对MHV68 ORF8抗原具有特异性：建立基础机制？DEC205和Vhh7依赖的抗原交叉呈递途径。2. 探索的能力？DEC205和VHH7加合物激活效应CD8 T细胞（或诱导它们从头产生）并建立对MHV68病毒的保护。如果成功的话，这些策略将为在不使用感染因子的情况下产生理想的T细胞反应打开大门，并通常改善免疫操作的选择，以达到治疗效果。！

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. ! PUBLIC HEALTH RELEVANCE: The DEC205 antibody, and an alpaca derived single domain antibody fragment, VHH7, will be engineered to receive, in a simple enzymatic reaction, an antigen of interest modified with chemical tags that will allow a detailed analysis of the attached antigenic cargo. The ability of such adducts to elicit a desirable CD8 T cell response against an infectious agent, MHV68, will be examined as a step towards developing this platform in a generalizable and practical vaccine strategy.