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

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)，它们被表达 并由精选的淋巴细胞如自然杀伤细胞(NK)和细胞毒性T细胞(CTL)有条件地分泌。基座 颗粒酶在很大程度上是基于A和B的数据，历史上一直被认为是促凋亡的效应物，因此它们 由NK和CTL在免疫突触与有问题的靶细胞(例如 癌细胞、病原体感染细胞)，并立即通过穿孔素进入细胞质，启动细胞死亡。 然而，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，在这个模型中 分泌的颗粒酶在细胞外空间发挥重要的信号功能，包括激活 巨噬细胞作为宿主防御的一部分。有趣的是，颗粒酶提呈和/或蛋白分解的失调 活动也可能是神经退行性或全身性疾病等衰弱疾病的病理生物学基础 自身免疫性疾病。因此，更充分地阐述颗粒酶的生物学是一个重要的未得到满足的需要，并且 需要技术来研究临床上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，我们称之为限制相互作用肽(RIP)来检测 颗粒酶在体内的蛋白降解活性与PET。从机制上讲，RIPs是作为一种非活性的前驱体给药的， 因此，目标内切酶对RIP的内部切割释放了放射性标记的多肽，该多肽 立即与附近的磷脂膜(即靶细胞的质膜)结合。 因此，内切酶附近放射性标记的裂解产物的积累提供了 感兴趣区域内酶活性的相对单位。作为初步数据，我们开发了64Cu-GB1，一种RIP 用来测量PET上颗粒酶B的活性。我们的概念验证数据显示64Cu-GB1检测到细胞毒 颗粒酶B活性池运输到致病靶细胞，但也是一个意想不到的非细胞毒池 作为对内毒素的炎症反应的一部分。在这个项目中，我们将在这些方面进行扩展 几个重要方向的数据。首先，我们将在目标1中测试64CuGB1是否可以用于颗粒酶B的研究 病原体刺激引起的自发免疫反应中的蛋白分解活性。这些数据将 对于理解这一方法学的效用至关重要，而不仅仅是检测药物增强 抗癌免疫反应。在Aim 2期间，我们将扩展RIP工具包以开发和研究探针 针对颗粒酶A和K的类胰蛋白酶蛋白分解活性，在最终目标中，我们将进行后期的 需要进行阶段性临床前实验，以判断使用RIP探针进行人体成像的可行性。作为一名模特 系统中，我们将研究我们的领先候选者64Cu-GB1。如果成功，这个项目将授予新的翻译 技术正处于一个关键的转折点，不再是经典的观点，而是简单地支持凋亡的效应器 人体免疫学和宿主防御病原体的多方面调节器。

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.