PET Probes Targeting Immune Cells for Imaging Tuberculosis

 描述(申请人提供)：结核病(TB)在全球范围内造成大量的发病率和死亡率。据估计，每年有900万新的活动性结核病病例，既包括原发结核病，也包括潜伏感染重新激活，导致130万人死亡。在感染结核分枝杆菌(Mtb)的人中，只有一小部分人(5%-10%)发展为活动性结核病，其余人是潜伏感染的。潜伏感染的人对分枝杆菌抗原表现出免疫应答，没有疾病的临床症状，但仍面临对活动性结核病重新激活的风险。艾滋病毒合并感染每年将重新激活的风险增加到10%。全球估计有20亿人(美国为1,120万人)潜伏感染是潜在的活动性、传染性结核病的重要宿主。由于缺乏有效的疫苗和耐药性的增加，在结核病流行的国家没有明确的控制和最终消除的途径。由于世界上三分之一的人口感染了结核分枝杆菌，迫切需要更好的疫苗、治疗方法和方法来确定那些具有最高再激活风险的人。结核病主要影响肺部，肺结核病变(肉芽肿)由淋巴细胞、激活的巨噬细胞和中性粒细胞以及结核杆菌组成。结核肉芽肿在细胞组成方面是不同的，甚至在同一个人中也是如此，并可能影响抗生素有效消毒个别病变的能力。这种皮损的异质性在我们的食蟹猴结核病模型中得到了概括。虽然[18F]标记的2-脱氧-2-氟-D-葡萄糖(FDG)PET/CT成像有助于连续跟踪结核分枝杆菌感染和疾病，以及在猕猴模型和人类中监测对药物的反应，但它并不专门针对结核病肉芽肿中的主要免疫细胞。拥有针对结核病感染肺部的关键免疫细胞(巨噬细胞、T细胞和中性粒细胞)的药物将有助于区分病变的缓解和进展，这将在监测治疗反应以及缩短新型疫苗的临床前和临床试验和 化疗药物。我们推测，针对特定免疫细胞群的PET显像剂将提供更详细的关于结核分枝杆菌感染的食蟹猴肉芽肿组成的信息，例如中性粒细胞、T细胞和激活的巨噬细胞的水平，这对于早期诊断激活的疾病和评估药物治疗的反应是重要的。在目标1中，我们将通过a)用FDG和64Cu-LLP2A对整个疾病过程中感染的猕猴进行连续成像，以及b)通过免疫组织化学和流式细胞术结合FDG成像，在尸检前注射染料-LLP2A，以确定结核分枝杆菌感染的猕猴结核肉芽肿中摄取VLA-4靶向示踪剂的机制。目标2将通过a)开发18F标记的趋化因子受体2型(CCR2)小分子拮抗剂；以及b)用18F标记的甘露糖对感染结核分枝杆菌的猕猴进行成像，从而开发专门针对巨噬细胞的放射性药物。在目标3中，我们将通过a)在小鼠炎症模型中筛选探针以提高靶向性并减少通过肝脏的清除量；以及b)测试放射性和染料cFlF探针在感染结核杆菌的猕猴中性粒细胞中的摄取，从而优化基于64Cu标记的cFlF多肽的中性粒细胞特异性探针。对于目标4，我们将研究在AIMS 1-3中开发的两到三种最有希望的示踪剂，以监测一种化疗(抗生素)方案之前、期间和之后猕猴结核病病变的细胞组成，以确定示踪剂摄取是否与皮损组织学和药物消毒个别病变的效率相关。

 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.