PET Probes Targeting Immune Cells for Imaging Tuberculosis

 描述（由申请人提供）：结核病（TB）是世界范围内发病率和死亡率较高的疾病。据估计，每年有900万新的活动性结核病例，包括原发性结核和潜伏感染的重新激活，导致130万人死亡。只有一小部分（5-10%）感染结核分枝杆菌（Mtb）的个体发展为活动性TB，其余为潜伏性感染。潜伏感染者对分枝杆菌抗原表现出免疫应答，没有疾病的临床症状，但仍有重新激活为活动性结核病的风险。艾滋病毒合并感染使再激活风险增加到每年10%。全球估计有20亿人（美国有1120万人）潜伏感染者是潜在的活动性传染性结核病的重要储存者。由于缺乏有效的疫苗和耐药性的日益普遍，在结核病流行的国家，没有明确的控制和最终消灭结核病的途径。由于世界上三分之一的人口感染了结核分枝杆菌，因此迫切需要更好的疫苗、治疗方法和方法来确定那些处于最高再活化风险的人。TB主要影响肺，其中形成由淋巴细胞、活化的巨噬细胞和嗜中性粒细胞以及Mtb杆菌组成的结核病损（肉芽肿）。TB肉芽肿在细胞组成方面是异质的，即使在同一个体内也是如此，并且可能影响抗生素有效地消毒个体病变的能力。这种病变异质性在我们的食蟹猴TB模型中重现。虽然[18 F]标记的2-脱氧-2-氟-D-葡萄糖（FDG）PET/CT成像有助于连续跟踪Mtb感染和疾病，以及监测猕猴模型和人类对药物的反应，但它并不特异性靶向作为TB肉芽肿中主要群体的免疫细胞。具有靶向TB感染的肺中存在的关键免疫细胞（巨噬细胞、T细胞和嗜中性粒细胞）的药剂将有助于区分正在消退的病变和正在进展的病变，这在监测治疗反应以及缩短新型疫苗和疫苗的临床前和临床试验方面将是非常宝贵的。 化疗药物我们假设，靶向特定免疫细胞群体的PET成像剂将提供有关结核分枝杆菌感染食蟹猴肉芽肿组成的更详细信息，如中性粒细胞、T细胞和活化巨噬细胞的水平，这些对于活化疾病的早期诊断和评估对药物治疗的反应至关重要。在目的1中，我们将通过以下方式确定64 Cu-LLP 2A（一种VLA-4靶向示踪剂）在Mtb感染的猕猴的TB肉芽肿中的摄取机制：a）在整个疾病期间用FDG和64 Cu-LLP 2A对感染的猕猴进行连续成像;和B）在尸检前注射染料-LLP 2A缀合物，以通过免疫组织化学和流式细胞术将免疫细胞定位与FDG成像相关联。目的2将通过以下方式开发特异性靶向巨噬细胞的放射性药物：a）开发趋化因子受体2型（CCR 2）的18 F标记的小分子拮抗剂;和B）用18 F标记的甘露糖对Mtb感染的猕猴进行成像。在目的3中，我们将通过以下方式优化基于64 Cu标记的cFlFlF基肽的嗜中性粒细胞特异性探针：a）在炎症小鼠模型中筛选探针以改善靶向并减少通过肝脏的清除;和B）测试放射性和染料cFlFlF探针在Mtb感染的猕猴的嗜中性粒细胞中的摄取。对于目标4，我们将研究目标1-3中开发的两到三种最有前途的示踪剂，以监测一种化疗（抗生素）方案之前、期间和之后猕猴TB病变的细胞组成，以确定示踪剂摄取是否与病变组织学和药物对单个病变的杀菌效率相关。

 DESCRIPTION (provided by applicant): Tuberculosis (TB) is responsible for substantial morbidity and mortality worldwide. It is estimated that there are 9 million new cases of active TB every year, representing both primary TB and reactivation of latent infection, resulting in 1.3 million deaths. Only a small fraction (5-10%) of individuals infected with Mycobacterium tuberculosis (Mtb) develop active TB, with the rest being latently infected. Persons with latent infection exhibit immune responsiveness to mycobacterial antigens and no clinical signs of disease but remain at risk of reactivation to active TB. HIV co-infection increases the reactivation risk to 10% annually. The estimated 2 billion people worldwide (11.2 million in the U.S.) with latent infection are a significant reservoir of potential active, infectious TB. Due to he lack of an effective vaccine and increasing prevalence of drug resistance, there is no clear path for control, and ultimately elimination, in countries where TB is endemic. Since one third of the world's population is infected with Mtb, there is a critical need for better vaccines, treatments, and methods to determine those at highest risk of reactivation. TB predominately affects the lungs where tuberculosis lesions (granulomas) form that consist of lymphocytes, activated macrophages and neutrophils, as well as Mtb bacilli. TB granulomas are heterogeneous in terms of cellular composition, even within the same individual and may influence the ability of antibiotics to effectively sterilize individual lesions. This lesional heterogeneity is recapitulatd in our cynomolgus macaque model of TB. While [18F]-labeled 2-deoxy-2-fluoro- D-glucose (FDG) PET/CT imaging has been helpful in serially tracking Mtb infection and disease, as well as in monitoring the response to drugs in the macaque model and in humans, it does not specifically target the immune cells that are the primary populations in TB granulomas. Having agents that target key immune cells present in TB-infected lungs (macrophages, T cells, and neutrophils) will help differentiate resolving from progressing lesions, which will be invaluable in monitoring therapeutic responses as well as shortening pre- clinical and clinical trials of novel vaccines and chemotherapeutics. We hypothesize that PET imaging agents targeting specific populations of immune cells will provide more detailed information about the composition of granulomas in Mtb-infected cynomolgus macaques, such as the levels of neutrophils, T cells and activated macrophages that are important for early diagnosis of activated disease and assessing the response to drug treatment. In Aim 1, we will determine the mechanism of uptake for 64Cu-LLP2A, a VLA-4 targeted tracer, in TB granulomas of Mtb-infected macaques by a) serially imaging of infected macaques throughout disease with FDG and 64Cu-LLP2A; and b) injecting dye-LLP2A conjugates pre-necropsy to correlate immune cell localization by immunohistochemistry and flow cytometry with FDG imaging. Aim 2 will develop radiopharmaceuticals that specifically target macrophages by a) developing 18F-labeled small molecule antagonists of chemokine receptor type 2 (CCR2); and b) imaging Mtb-infected macaques with 18F-labeled mannose. In Aim 3 we will optimize neutrophil-specific probes based on 64Cu-labeled cFlFlF-based peptides by a) screening probes in a mouse model of inflammation to improve targeting and reduce clearance through the liver; and b) testing the uptake of radio- and dye-cFlFlF probes in neutrophils of Mtb-infected macaques. For Aim 4 we will investigate two to three of the most promising tracers developed in Aims 1-3 to monitor the cellular composition of TB lesions in macaques before, during, and after one chemotherapeutic (antibiotic) regimen, to determine if tracer uptake correlates with lesional histology and the efficiency of drugs to sterilize individual lesions.