Biomolecular probes for imaging protease activities

描述(申请人提供)：通过非侵入性成像及早发现小肿瘤具有显著改善癌症患者存活率和改善生活质量的潜力。然而，早期检测目前仅限于检测由大约10亿个肿瘤细胞组成的肿块。要提高这一十亿细胞的阈值，将需要一些方法来提高对肿瘤的分子显像剂的选择性，并避免正常组织对显像剂的摄取。为了减少背景和提高成像对比度，提出了一种分子显像剂，其中肿瘤摄取将通过已知在肿瘤中具有选择性活性的酶的作用而被放大。为了实现这一点，我们将首先鉴定并亲和成熟对乳腺肿瘤和其他肿瘤类型中存在的受体具有高亲和力和特异性的抗体片段。然后使用蛋白质工程方法对抗体进行修饰，使结合活性依赖于蛋白酶活性，从而产生蛋白酶激活的抗体。同时，我们将使用蛋白质展示技术来识别显示活性、选择性和稳定性所需属性的蛋白酶底物，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显示人和小鼠肿瘤受体同系物之间的交叉反应，以允许快速转移到临床的可能性。在小鼠异种移植模型中，将通过近红外成像来评估由蛋白酶激活的抗体带来的选择性增加和成像对比度的增加。这项工作有望产生高靶向性、蛋白酶活性的成像探针，这些探针对检测多种肿瘤类型具有更高的选择性，并可能使更早的肿瘤检测或肿瘤表型鉴定成为可能。更广泛地说，这个项目将展示一种新的方法学方法，可以广泛应用于各种成像应用中提高对比度。)

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. )