Biomolecular probes for imaging protease activities

描述（由申请人提供）：通过非侵入性成像对小肿瘤的早期检测具有显著提高癌症患者生存率和提高生活质量的潜力。然而，早期检测目前仅限于检测由超过10亿个肿瘤细胞组成的肿块。要达到这个十亿细胞的临界值，就需要将分子显像剂以更高的选择性输送到肿瘤中，并避免显像剂在正常组织中的吸收。为了减少背景和提高成像对比度，提出了一种分子显像剂，其中肿瘤摄取将被肿瘤中已知的选择性活性酶的作用放大。为了实现这一目标，我们将首先鉴定和亲和力成熟的抗体片段具有高亲和力和特异性的受体存在于乳腺肿瘤和其他肿瘤类型。然后使用蛋白质工程方法修饰抗体，使结合活性依赖于蛋白酶活性，从而产生蛋白酶活化的抗体。同时，我们将使用蛋白质展示技术来鉴定蛋白酶底物，这些底物表现出必要的活性、选择性和稳定性，以实现精确的体内靶向。蛋白酶激活的抗体成像探针将被设计成在人类和小鼠肿瘤受体同源物之间表现出交叉反应性，从而允许快速转化为临床的可能性。在小鼠异种移植物模型中，将通过近红外成像来评估蛋白酶激活抗体所带来的选择性和成像对比度的增加。这项工作有望产生高度靶向的蛋白酶活性成像探针，这种探针在检测多种肿瘤类型方面表现出更高的选择性，并且可以实现早期肿瘤检测或肿瘤表型。更一般地说，这个项目将展示一种新的方法方法，可以广泛地应用于提高各种成像应用的对比度。

DESCRIPTION (provided by applicant): Early detection of small tumors by non-invasive imaging holds the potential to substantially improve cancer patient survival and improve quality of life. However, early detection is currently limited to detecting masses composed of more than about one billion tumor cells. Improving upon this billion-cell threshold will require methods to deliver molecular imaging agents to tumors with increased selectivity, and to avoid the uptake of imaging agents in normal tissues. To reduce background and improve imaging contrast, a molecular imaging agent is proposed wherein tumor uptake will be amplified by the action of enzymes known to be selectively active in tumors. To accomplish this, we will first identify and affinity matures an antibody fragment with high affinity and specificity for a receptor present in breast tumors and other tumor types. The antibody will then be modified using protein engineering methods render binding activity dependent upon protease activity thereby generating protease- activated antibodies. In parallel, we will use protein display technologies to identify protease substrates that exhibit the requisite properties of activity, selectivity, and stability to enable precise in vivo targeting. Protease-activated antibody imaging probes will be designed to exhibit cross reactivity between human and mouse tumor receptor homologs to allow for the possibility of rapid translation to the clinic. The increase in selectivity and resultant increase in imaging contrast conferred by protease-activated antibodies will be assessed in mouse xenograft models by near-infrared imaging. This work is expected to yield highly-targeted, protease-activity imaging probes that exhibit increased selectivity for detecting multiple tumor types, and that could enable earlier tumor detection, or tumor phenotyping. More generally, this project will demonstrate a novel methodological approach that could be broadly applied to improve contrast in a variety of imaging applications. )