Enzymatic modification of anti-DEC205 to manipulate its immunogenic properties

酶促修饰抗 DEC205 以操纵其免疫原性特性

• 批准号: 8386128
• 负责人: Hidde L. Ploegh
• 金额: $ 29.25万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386128
• 关键词: Address; Adjuvant; Alpaca; Antibodies; Antigen Targeting; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Binding; Biochemical Reaction; Biochemistry; Biological; Biotin; CD8B1 gene; Cell Fractionation; Cell-Matrix Junction; Cells; Chemicals; Chemistry; Cross Presentation; Dendritic Cells; Dose; Engineering; Epitopes; Generations; Genetic; Goals; Herpesviridae; Immune; Immune response; Immunochemistry; Immunoglobulin Fragments; In Vitro; Infectious Agent; Lead; MHC Class I Genes; MHC Class II Genes; Membrane Proteins; Methods; Microscopy; Modification; Monitor; Monoclonal Antibodies; Mouse Protein; Mus; Names; Outcome; Peptides; Phase; Positioning Attribute; Procedures; Property; Proteins; Reaction; Regimen; Reporting; Role; Route; Site; Specificity; Staphylococcus aureus; T cell response; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; Therapeutic Effect; Toxoplasma gondii; Vaccination; Vaccines; Virus; adduct; antibody conjugate; comparative efficacy; cost effective; cytotoxic; flexibility; fluorophore; immunogenic; improved; in vivo; interest; pathogen; somatic cell nuclear transfer; sortase; targeted delivery; tool

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.

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。! 公共卫生关系：DEC 205抗体和源自羊驼的单结构域抗体片段VHH 7将被工程化以在简单的酶促反应中接收用化学标签修饰的感兴趣的抗原，所述化学标签将允许对所附接的抗原进行详细分析。 抗原货物这种加合物引发针对感染因子MHV 68的期望的CD 8 T细胞应答的能力将作为在可推广和实用的疫苗策略中开发该平台的一个步骤进行检查。