Novel probes for near-infrared fluorescence imaging-guided oncologic surgery

 描述(申请人提供)：肿瘤外科手术的结果关键取决于完全切除肿块，以防止肿瘤再生长和疾病复发。这是一项具有挑战性的任务，特别是在侵袭性和播散性疾病的病例中，肿瘤边缘模糊，微观肿瘤往往无法与周围正常组织区分开来。术前诊断成像提供了有关肿瘤位置和大小的丰富信息。然而，在手术过程中，外科医生主要通过对手术区域的肉眼检查和触诊来识别肿瘤和肿瘤边缘；结果，肿瘤往往没有被发现，肿瘤边缘被错误地确定。另一方面，过度切除肿瘤边缘往往会导致严重的术后并发症。使用近红外荧光(NIRF)实现的术中肿瘤实时可视化可以解决这些问题，这将使外科医生能够定位和切除肉眼无法看到的肿瘤，并能够更精确地确定肿瘤边缘。这个试点项目的目的是通过在肿瘤动物模型上开发和测试新一代肿瘤靶向的NIRF探针，来证明术中灵敏和准确的肿瘤可视化的可行性。我们的方法是基于这样一个假设，即这种先进的探针可以使用一类新型的合理设计的分子-设计的锚蛋白重复蛋白(DARPins)-作为近红外荧光团的载体来开发。由于其体积小、高亲和力和对肿瘤生物标记物的特异性，DARPins在肿瘤中快速高效地积聚，并从非靶组织中清除。DARPins很容易在细菌中生产和纯化；它们可以很容易地进行基因改造，以实现与荧光团精确的、特定部位的结合。DARPins的高度稳定的核心结构导致了人体血液的长期稳定。这些特性使DARPins成为抗体(Ab)、Ab衍生物、多肽和小分子的极佳替代品，后者传统上用于成像探针开发。我们将通过追求两个特定的目标来验证我们的假设并实现我们的目标：特定的目标1.体外开发和表征针对人表皮生长因子受体2(Her2)的基于DARPin的NIRF探针。具体目的2.在卵巢癌小鼠模型中，通过使用DARPin衍生的NIRF探针，证明术中显示播散性肿瘤的可行性。这一可行性项目的结果将对癌症治疗产生重大积极影响，使手术切除肿瘤更有效率，减少手术后发病率，从而延长和挽救人类生命。

 DESCRIPTION (provided by applicant): The outcomes of oncologic surgery critically depend on complete removal of the tumor mass, which prevents tumor regrowth and disease reoccurrence. This is a challenging task, especially in cases of invasive and disseminated disease in which tumor margins are poorly defined and microscopic tumors are often indistinguishable from surrounding normal tissues. Preoperative diagnostic imaging provides a wealth of information on the tumors' location and size. However, during surgery, surgeons identify tumors and tumor margins largely by visual inspection of the surgical field and by palpation; as a result, tumors often remain undetected, and tumor margins are determined incorrectly. On the other hand, excessive resection of tumor margins often leads to severe postoperative morbidity. These problems may be solved using real- time intraoperative visualization of tumors enabled by near-infrared fluorescence (NIRF), which would give a surgeon the ability to locate and remove the tumors that cannot be seen by the naked eye and enable more precise determination of tumor margins. The objective of this pilot project is to demonstrate the feasibility of sensitive and accurate intraoperative tumor visualization by developing and testing in an animal model of cancer a new generation of tumor-targeted NIRF probes. Our approach is based on the hypothesis that such advanced probes can be developed using a novel class of rationally engineered molecules-designed ankyrin repeat proteins (DARPins)-as carriers of NIR fluorophores. Owing to their small size, high affinity and specificity for tumor biomarkers, DARPins rapidly and efficiently accumulate in tumors and clear from nontarget tissues. DARPins are easy to produce in bacteria and purify; they can be easily modified genetically to enable precise, site-specific conjugation with fluorophores. DARPins' highly stable core structure results in prolonged stability in human blood. These properties make DARPins an excellent alternative to antibodies (Ab), Ab derivatives, peptides, and small molecules, which are traditionally used for imaging probe development. We will test our hypothesis and achieve our objective by pursuing two specific aims: Specific Aim 1. Develop and characterize in vitro DARPin-based NIRF probes that target human epidermal growth factor receptor 2 (Her2). Specific Aim 2. Demonstrate the feasibility of intraoperative visualization of disseminated tumors by using DARPin-derived NIRF probes in a mouse model of ovarian cancer. The outcomes of this feasibility project will have a significant positive impact on cancer management by making surgical resection of tumors more efficient and reducing post-surgical morbidity, thereby prolonging and saving human life.