New radiotracer development to study immune cell mobilization of granzyme proteolytic activity

开发新的放射性示踪剂来研究免疫细胞动员颗粒酶蛋白水解活性

基本信息

批准号：
10591415
负责人：
Charles Scott Craik
金额：
$ 66.04万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-20 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10591415
关键词：
Amino Acids Animal Model Apoptotic Bacteria Bacterial Infections Biochemistry Biodistribution Biological Assay Biological Models Biology Bypass Cell Death Cell membrane Cells Coculture Techniques Communicable Diseases Complex Cytokine Activation Cytosol Cytotoxic T-Lymphocytes Data Development Disease Drug Kinetics Endotoxins Enzymes Escherichia coli Extracellular Space Fluorescence Resonance Energy Transfer Goals Granzyme Host Defense Human Human Activities Image Immune Immune response Immune system Immunology In Vitro Inflammation Inflammatory Response Intercept Klebsiella pneumoniae Knockout Mice Listeria monocytogenes Lymphocyte Macrophage Activation Masks Mass Spectrum Analysis Measurement Measures Membrane Methodology Modeling Mouse Strains Mus Mycobacterium marinum Mycobacterium tuberculosis Myositis Nature Nerve Degeneration Organism Pathogenicity Peptide Hydrolases Peptides Pharmaceutical Chemistry Phospholipids Positron-Emission Tomography Productivity Proteolysis Pseudomonas aeruginosa Qualifying Radioactive Radiolabeled Research Resolution Rodent Role Safety Serine Protease Signal Transduction Specificity Staphylococcus aureus Technology Testing Time Tryptase Viral Pneumonia Virus Diseases Xenobiotics analog anti-cancer cancer cell clinical application clinically relevant clinically significant combat cytotoxic design dosimetry empowerment enzyme activity experimental study extracellular human imaging imaging approach imaging probe imaging study immunological synapse in vivo in vivo imaging interest lead candidate novel nuclear imaging optical imaging pathogen pathogenic virus perforin pharmacologic pre-clinical preclinical study radiochemical radiotracer response systemic autoimmune disease tomography tool trafficking

Amino Acids Animal Model Apoptotic Bacteria Bacterial Infections Biochemistry Biodistribution Biological Assay Biological Models Biology Bypass Cell Death Cell membrane Cells Coculture Techniques Communicable Diseases Complex Cytokine Activation Cytosol Cytotoxic T-Lymphocytes Data Development Disease Drug Kinetics Endotoxins Enzymes Escherichia coli Extracellular Space Fluorescence Resonance Energy Transfer Goals Granzyme Host Defense Human Human Activities Image Immune Immune response Immune system Immunology In Vitro Inflammation Inflammatory Response Intercept Klebsiella pneumoniae Knockout Mice Listeria monocytogenes Lymphocyte Macrophage Activation Masks Mass Spectrum Analysis Measurement Measures Membrane Methodology Modeling Mouse Strains Mus Mycobacterium marinum Mycobacterium tuberculosis Myositis Nature Nerve Degeneration Organism Pathogenicity Peptide Hydrolases Peptides Pharmaceutical Chemistry Phospholipids Positron-Emission Tomography Productivity Proteolysis Pseudomonas aeruginosa Qualifying Radioactive Radiolabeled Research Resolution Rodent Role Safety Serine Protease Signal Transduction Specificity Staphylococcus aureus Technology Testing Time Tryptase Viral Pneumonia Virus Diseases Xenobiotics analog anti-cancer cancer cell clinical application clinically relevant clinically significant combat cytotoxic design dosimetry empowerment enzyme activity experimental study extracellular human imaging imaging approach imaging probe imaging study immunological synapse in vivo in vivo imaging interest lead candidate novel nuclear imaging optical imaging pathogen pathogenic virus perforin pharmacologic pre-clinical preclinical study radiochemical radiotracer response systemic autoimmune disease tomography tool trafficking

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项目摘要

Project Abstract: The human granzymes are a somewhat mystical class of five serine proteases (A, B, H, K, M) that are expressed and conditionally secreted by select lymphocytes like natural killer (NK) and cytotoxic T cells (CTL). Based largely on data for A and B, granzymes have been historically regarded as pro-apoptotic effectors whereby they are presented transiently by NK and CTLs at the immunological synapse with a problematic target cell (e.g. cancer cell, pathogen infected cell), and immediately shuttled into the cytosol via perforin to initiate cell death. However, emerging data has begun to challenge this canon and present a more complex model in which secreted granzymes perform essential signaling functions in extracellular space, including activation of macrophages as part of host defense. Interestingly, dysregulation of granzyme presentation and/or proteolytic activity may also underlie the pathobiology of debilitating diseases like neurodegenerative or systemic autoimmune diseases. Thus, more fully elaborating the biology of granzymes is an important unmet need, and requires technologies to study granzymes in the most clinically relevant animal models and humans themselves. To this end, we developed a novel imaging approach we term “restricted interaction peptides” (RIP) to detect granzyme proteolytic activity in vivo with PET. Mechanistically, RIPs are administered as an inactive pro-form, whereupon internal cleavage of the RIP by the target endoprotease releases a radiolabeled peptide that immediately associates with nearby phospholipid membranes (i.e. the plasma membrane of the target cell). Thus, accumulation of the radiolabeled cleavage product adjacent to the endoprotease provides a readout of the relative units of enzyme activity within a region of interest. As preliminary data, we developed 64Cu-GB1, a RIP that measures granzyme B activity on PET. Our proof of concept data show that 64Cu-GB1 detects the cytotoxic pool of granzyme B activity trafficking to pathogenic target cells, but also an unexpected non-cytotoxic pool elicited as part of an inflammatory response to an endotoxin. During this project, we will expand upon these data in several important directions. First, we will test in Aim 1 if 64Cu-GB1 can be applied to study granzyme B proteolytic activity in the spontaneous immune responses arising due to pathogen stimulation. These data will be crucial to understanding the utility of this methodology beyond simply detecting pharmacologically augmented anticancer immune responses. During Aim 2, we will expand the RIP toolkit to develop and study probes targeting the tryptase proteolytic activities of granzymes A and K. During the final Aim, we will carry out the late stage preclinical experiments required to judge the feasibility of human imaging with RIP probes. As a model system, we will study our lead candidate 64Cu-GB1. If successful, this project will confer new translational technologies at a crucial inflection point away from the classical view as simply pro-apoptotic effectors toward multifaceted regulators of human immunology and host defense against pathogens.

项目摘要：人类颗粒是一种有点神秘的类别，是五个连环蛋白酶（a，b，h，k，m）的表达并由精选的淋巴细胞（如天然杀手（NK）和细胞毒性T细胞（CTL））分泌。基于在很大程度上，在A和B的数据上，谷物酶在历史上被认为是促凋亡的效果由NK和CTL在免疫突触中暂时呈现，并具有有问题的靶细胞（例如，癌细胞，病原体感染细胞），并立即通过穿孔蛋白将细胞溶胶穿梭到细胞溶胶中，以启动细胞死亡。但是，新兴数据已经开始挑战这一经典，并提出了一个更复杂的模型分泌的颗粒酶在细胞外空间中执行必不可少的信号传导功能，包括激活巨噬细胞作为东道国防御的一部分。有趣的是，颗粒酶表示和/或蛋白水解的失调活动还可能是神经退行性或全身性疾病的衰弱疾病的病理生物学的基础自身免疫性疾病。这更充分地阐述了颗粒酶的生物学是一个重要的未满足需求，并且需要技术来研究最临床相关的动物模型和人类本身中的颗粒酶。为此，我们开发了一种新型的成像方法，我们称为“限制性相互作用胡椒粉”（RIP）以检测用PET在体内颗粒酶蛋白水解活性。从机械上讲，撕裂是作为非活动形式的施用的因此，靶内预防酶对RIP的内部切割释放了放射性标记的肽理想情况下，与附近的磷脂膜相关联（即靶细胞的质膜）。那是内脏蛋白酶附近的放射标记的切割产品的积累，提供了读数酶活性的相对单位在感兴趣的区域内。作为初步数据，我们开发了64CU-GB1，RIP 这测量了PET上的粒酶B活性。我们的概念证明数据表明，64CU-GB1检测到细胞毒性颗粒酶B活性运输到致病靶细胞上，但也是意外的无毒性池作为对内毒素的炎症反应的一部分引起的。在这个项目中，我们将扩展这些数据在多个重要方向上。首先，如果可以应用64CU-GB1，我们将在AIM 1中进行测试由于病原体刺激引起的赞助者免疫反应中的蛋白水解活性。这些数据将除了简单地检测药物增强外，对理解这种方法的实用性至关重要抗癌免疫反应。在AIM 2期间，我们将扩展RIP工具包以发展和研究问题针对粒酶A和K的胰蛋白酶蛋白水解活性。在最终目标中，我们将执行最新的判断人类成像的可行性所需的阶段临床前实验。作为模型系统，我们将研究主要候选人64CU-GB1。如果成功，该项目将召集新的翻译至关重要的拐点处的技术远离经典视图，因为简单地促凋亡效应人类免疫学的多方面调节剂和托管防御病原体的防御。