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，可用于将抗原靶向DC：通过化学结合或基因融合将抗原部分附着到？DEC205上，使其能够输送到DC。这会产生一种有效的适应性免疫反应，包括针对附着在DEC205上的抗原的CD4和CD8T细胞。这种方法以及针对树突状细胞上其他表面蛋白的其他抗体的类似修改，如第二类MHC产品，有望成为一种可能的疫苗策略。大多数化学偶联方法的精密度不足，以及将感兴趣的有效载荷结合到DEC205上并提纯所产生的加合物所需的劳动密集型基因融合方法，这些都表明了一种消除这些缺点的替代方法。一种高效和直接的化学-酶替代方法，使用来自金黄色葡萄球菌的索糖酶，被提议用来详细分析？DEC205加合物理想的免疫原性的基础机制。将与Vhh7进行比较，Vhh7是一种来自羊驼的单区抗鼠II类MHC抗体。Vhh7以类似的方式被设计作为分类酶底物，通过产生荧光和生物素化的衍生物来验证，作为展示其特异性和与I类MHC产物紧密结合的手段。在DEC205重链的C末端引入LPXTG基序或在Vhh7上引入LPXTG基序允许以化学计量方式特异性地附着T细胞表位、所选择的荧光或生物素化的有效载荷，甚至蛋白质大小的取代基。这种被称为分选标记的程序，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)特异性地递送到体内的DEC205+和II类MHC+抗原提呈细胞，而不需要大量独立的基因？DEC205或Vhh7融合构建体。所提出的方法具有附加的优点，即可以安装非天然取代基，例如容易切割的连接体或佐剂，以提高这种加合物的效力，并且甚至可以使用点击化学方法进行C-端到C-端的融合，以比较C-N和C-C融合的呈现效果。这些目标将在两个特定的目标下实现，应用于CD8 T细胞，针对MHV68 ORF8抗原，从体细胞核移植产生的克隆小鼠获得：1.建立依赖于DEC205和Vhh7的抗原交叉呈递途径的机制。2.探讨？DEC205和VHH7加合物对效应CD8 T细胞的激活(或从头诱导)以及对MHV68病毒的保护作用。如果成功，这些策略将为在不使用感染剂的情况下创造令人满意的T细胞反应打开大门，并通常改善免疫操作的选择，以实现治疗效果。好了！ 公共卫生相关性：DEC205抗体和羊驼来源的单域抗体片段VHH7将经过改造，在简单的酶反应中获得感兴趣的抗原，并用化学标签进行修饰，从而对所附的 抗原性货物。这些加合物对感染剂MHV68诱导理想的CD8 T细胞反应的能力将被检验，作为在可推广和实用的疫苗策略中开发这一平台的一步。

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.