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

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一个非常理想但具有挑战性的目标。白细胞特异性单域抗体（VHHs）和基于sortase的酶偶联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，以跟踪免疫反应和成像炎症。酶修饰平台可以很容易地将18F、64Cu或68Ga特异位点快速安装到vhh上，这些vhh将从识别人类和小鼠抗原的面板中选择，包括II类MHC、CD3、CD4、CD8、CTLA4、PD-1和PD-L1。这些研究将包括优化标记效率和通过PET成像对主要候选药物进行药代动力学分析。我们将分析白细胞在正常小鼠、炎症诱导小鼠以及异种移植和同基因肿瘤模型中的分布。我们的初步数据显示产生了一个位点特异性的18f标记的单域抗体，用于小鼠II类MHC产物18F-VHH7。我们使用18F-VHH7对小鼠进行正电子发射断层扫描（PET），使用野生型，MHC II-/-和NOD-SCID小鼠异种移植了人类黑色素瘤细胞系。18F-VHH7不仅能迅速从循环中清除（1/2<20 min），而且还能显示二级淋巴器官，具有显著的特异性。此外，18F-VHH7可以清晰地显示与黑色素瘤异种移植相关的II类MHC+细胞，为早期无创检测炎症细胞和淋巴细胞作为疾病指标奠定了基础。我们提出以下具体目标：目标一。开发一种标记策略，生成18f标记的单域羊驼衍生抗体（VHHs），用于PET成像，对抗小鼠cd3 γ ε、CD11b和II类MHC。我们还将开发一种使用NOTA作为螯合剂，用放射性金属（如68Ga或64Cu）标记位点特异性蛋白质的方法。目标2。从重组小鼠CTLA4， CD4, CD8， PD-1和PD-L1免疫的羊驼噬菌体展示文库中分离单域抗体，以排序酶准备格式进行18F， 68Ga或64Cu标记。目标3。使用目标1和目标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基因肿瘤小鼠中的白细胞分布。这些目标的成功完成将为将这种方法转化为临床环境提供必要的基础。

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.