Non-invasive imaging of the immune response based on the use of isotopically labeled single domain antibody fragments

基于使用同位素标记的单域抗体片段的免疫反应的非侵入性成像

• 批准号: 8873207
• 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: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8873207
• 关键词: Alpaca; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antigens; Blood Circulation; CD3 Antigens; CD8B1 gene; CTLA4 gene; Cell Line; Cell Surface Proteins; Cells; Chelating Agents; Clinical; Data; Disease; Drug Kinetics; Foundations; Future; Generations; Goals; Hand; Health; Human; ITGAM gene; Image; Immune; Immune response; Immunoglobulin Fragments; Immunological Diagnosis; Inflammation; Inflammatory; Inflammatory Response; Isotopes; Label; Lead; Leukocytes; Libraries; Lymphocyte; Lymphoid; MHC Class II Genes; Melanoma Cell; Methods; Modeling; Modification; Mus; NOD/SCID mouse; Non-Invasive Cancer Detection; Organ; Phage Display; Phase; Phase II Clinical Trials; Positron-Emission Tomography; Proteins; Radioisotopes; Radiolabeled; Reaction; Reagent; Recombinants; Reporting; Resolution; Site; Specificity; Staging; T-Lymphocyte; Translating; Translations; Xenograft procedure; base; clinical application; clinically relevant; imaging agent; macrophage; melanoma; mouse model; non-invasive imaging; novel; novel diagnostics; radiotracer; research study; single photon emission computed tomography; sortase; tool; tumor

DESCRIPTION (provided by applicant): Non-invasive imaging of an inflammatory response or of an immune response against a tumor remains a highly desirable yet challenging goal. A combination of leukocyte-specific single-domain antibodies (VHHs) and a sortase-based enzymatic conjugation reaction will be used for the creation of immunodiagnostics suitable for positron emission tomography (PET) to track immune response and to image inflammation. The enzymatic modification platform readily allows the site-specific and rapid installation of 18F, 64Cu or 68Ga onto VHHs that will be chosen from a panel recognizing human and mouse antigens, including Class II MHC, CD3, CD4, CD8, CTLA4, PD-1 and PD-L1. These studies will involve optimization of labeling efficiency and pharmacokinetic analysis by PET imaging of lead candidates. We shall analyze the distribution of leukocytes in normal mice, in mice in which inflammation is induced, and in xenografted and syngeneic tumor models. Our preliminary data show the generation of a site-specifically 18F-labeled single domain antibody specific for murine Class II MHC products, 18F-VHH7. We used 18F-VHH7 to perform positron emission tomography (PET) in mice, using wild type, MHC II-/- and NOD-SCID mice xenografted with a human melanoma cell line. Not only is 18F-VHH7 rapidly cleared from the circulation, (t 1/2<20 min), it also reveals secondary lymphoid organs with remarkable specificity. Moreover, Class II MHC+ cells associated with the melanoma xenograft were clearly visualized with 18F-VHH7, setting the stage for early non-invasive detection of inflammatory cells and lymphocytes as an indicator of disease. We propose the following specific aims: Aim 1. Develop a labeling strategy to generate 18F-labeled single domain alpaca-derived antibodies (VHHs) against mouse CD3γϵ, CD11b and Class II MHC for use in PET imaging. We will also develop a method for site-specific protein labeling with radiometals such as 68Ga or 64Cu, using NOTA as chelating agent. Aim 2. Isolate single domain antibodies from a phage display library obtained from an alpaca immunized with recombinant mouse CTLA4, CD4, CD8, PD-1 and PD-L1 in a sortase-ready format to enable labeling with 18F, 68Ga or 64Cu. Aim 3. Using the tools developed in Aims 1 and 2 perform PET imaging to explore the distribution of leukocytes in normal mice, in animals in which inflammation is induced, and in mice with xenografted and syngeneic tumors. The successful completion of these aims will provide the necessary foundation for translation of this approach to a more clinical setting.

描述（由申请人提供）：炎症反应或针对肿瘤的免疫反应的非侵入性成像仍然是非常理想但具有挑战性的目标。白细胞特异性单域抗体（VHH）和基于分选酶的酶促缀合反应的组合将用于创建适合于正电子发射断层扫描（PET）的免疫诊断，以跟踪免疫反应和炎症成像。酶促修饰平台容易地允许将18F、64 Cu或68 Ga位点特异性地和快速地安装到VHH上，所述VHH将选自识别人和小鼠抗原的组，包括II类MHC、CD 3、CD 4、CD 8、CTLA 4、PD-1和PD-L1。这些研究将涉及标记效率的优化和通过PET成像的主要候选药物的药代动力学分析。我们将分析白细胞在正常小鼠、诱导炎症的小鼠以及异种移植和同源肿瘤模型中的分布。我们的初步数据显示，产生的位点特异性18 F标记的单域抗体特异性的小鼠II类MHC产品，18 F-VHH 7。我们使用18F-VHH 7在小鼠中进行正电子发射断层扫描（PET），使用野生型、MHC II-/-和NOD-SCID小鼠异种移植人黑色素瘤细胞系。18F-VHH 7不仅从循环中迅速清除（t1/2<20 min），还显示出具有显著特异性的次级淋巴器官。此外，与黑素瘤异种移植物相关的II类MHC+细胞用18F-VHH 7清楚地可视化，为炎症细胞和淋巴细胞作为疾病指标的早期非侵入性检测奠定了基础。我们提出以下具体目标：目标1。开发一种标记策略，以产生18F标记的抗小鼠CD 3 γ γ A、CD 11b和II类MHC的单结构域羊驼衍生抗体（VHH），用于PET成像。我们还将开发一种方法，用于位点特异性的蛋白质标记与放射性金属，如68 Ga或64 Cu，使用NOTA作为螯合剂。目标2.从获自羊驼的噬菌体展示文库中分离单结构域抗体，所述羊驼用重组小鼠CTLA 4、CD 4、CD 8、PD-1和PD-L1以分选酶就绪形式免疫以能够用18F、68 Ga或64 Cu标记。目标3.使用目标1和2中开发的工具进行PET成像，以探索正常小鼠、诱导炎症的动物以及异种移植和同源肿瘤小鼠中白细胞的分布。这些目标的成功完成将为将这种方法转化为更临床的环境提供必要的基础。