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

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：人类颗粒酶是一类有点神秘的五种丝氨酸蛋白酶（A、B、H、K、M），它们表达并由自然杀伤细胞 (NK) 和细胞毒性 T 细胞 (CTL) 等特定淋巴细胞有条件地分泌。主要基于 A 和 B 的数据，颗粒酶历来被视为促凋亡效应物，因此它们 NK 和 CTL 在免疫突触处短暂呈递有问题的靶细胞（例如癌细胞，病原体感染的细胞），并立即通过穿孔素穿梭到细胞质中启动细胞死亡。然而，新兴数据已经开始挑战这一准则，并提出了一个更复杂的模型，其中分泌的颗粒酶在细胞外空间执行重要的信号传导功能，包括激活巨噬细胞作为宿主防御的一部分，故意调节颗粒酶的表达和/或蛋白水解。活动也可能是神经退行性疾病或系统性衰弱性疾病的病理学基础因此，更全面地阐述颗粒酶的生物学是一个重要的未满足的需求。需要技术来研究临床最相关的动物模型和人类本身的颗粒酶。为此，我们开发了一种新的成像方法，我们称之为“限制性相互作用肽”（RIP）来检测 PET 体内颗粒酶的蛋白水解活性从机制上讲，RIP 以无活性的前体形式施用。随后，目标内切蛋白酶对 RIP 进行内部切割，释放放射性标记的肽，该肽立即与附近的磷脂膜（即靶细胞的质膜）结合。因此，邻近内切蛋白酶的放射性标记裂解产物的积累提供了作为初步数据，我们开发了 64Cu-GB1，一种 RIP。测量 PET 上的颗粒酶 B 活性我们的概念验证数据表明 64Cu-GB1 可以检测到细胞毒性。转运至致病靶细胞的颗粒酶 B 活性池，也是一个意想不到的非细胞毒性池作为对内毒素的炎症反应的一部分而引起的。在这个项目中，我们将扩展这些。首先，我们将在目标1中测试64Cu-GB1是否可以用于研究颗粒酶B。由于病原体刺激而产生的自发免疫反应中的蛋白水解活性。除了简单地检测药理学增强之外，对于理解这种方法的实用性至关重要在目标 2 期间，我们将扩展 RIP 工具包来开发和研究探针。针对颗粒酶 A 和 K 的类胰蛋白酶蛋白水解活性。在最终目标期间，我们将进行后期判断使用 RIP 探针作为模型进行人体成像的可行性所需的阶段临床前实验。系统，我们将研究我们的主要候选材料 64Cu-GB1 如果成功，该项目将赋予新的转化能力。技术正处于一个关键的转折点，远离了简单的促凋亡效应器的经典观点人类免疫学和宿主防御病原体的多方面调节剂。