VEGF-based Targeted Imaging of Tumor Vasculature

描述(申请人提供)：该项目的总体目标是为肿瘤血管生成中血管内皮生长因子(VEGF)的成像受体开发有针对性的探针。过表达的血管内皮生长因子受体在肿瘤血管生成的发生和发展中起着关键作用，因此，这些受体是开发血管生成抑制剂的主要靶点。然而，这些受体没有被用作开处方或监测抗血管生成治疗的生物标志物，因为没有非侵入性的方法来评估它们的流行率。因此，血管内皮生长因子受体的非侵入性成像解决了未得到满足的临床需求，并可能导致对抗血管生成治疗患者的循证选择，以及个性化治疗方案的设计。此外，由于据报道，在恶性生长初期，肿瘤和邻近的宿主血管中都有血管内皮生长因子受体的过度表达，因此，血管内皮生长因子受体成像可能被用于早期诊断和鉴别良恶性病变。在这个项目的第一阶段，我们开发了适合于临床开发的基于血管内皮生长因子的新型探针，用于血管内皮生长因子受体的近红外荧光(NIRF)和单光子发射计算机断层扫描(SPECT)成像。将造影剂与适当设计的血管内皮生长因子进行定点结合，可产生与血管内皮细胞上的血管内皮生长因子受体结合的探针，与亲代血管内皮生长因子一样有效。我们证实，我们的基于血管内皮生长因子的探针经历了受体介导的内化，并在过度表达血管内皮生长因子受体的肿瘤和宿主内皮细胞中积聚。重要的是，使用失活的基于血管内皮生长因子的探针，失去了与血管内皮生长因子受体结合的能力，我们评估了非特异性机制对探针在肿瘤血管中聚集的贡献。我们的靶向成像探针是基于一种新型的单链(Sc)血管内皮生长因子，相对于通过二硫键连接的两个亚单位的传统血管内皮生长因子，它在表达、复性和纯化方面都有显著改进。通过我们专有的人源化半胱氨酸标签(Cys-Tag)，对比剂与scVEGF进行特定部位的结合，确保了成像探针的均一性。综上所述，我们的第一阶段结果为基于血管内皮生长因子的探针的临床开发开辟了道路，用于肿瘤血管中血管内皮生长因子受体的选择性和特异性成像。在第二阶段，我们将建立基于单链血管内皮生长因子的探针的安全性，开发可扩展的GLP结合物生产，并表征这些探针用于预测/监测对特定的血管内皮生长因子受体抑制剂的反应和早期诊断肺部恶性病变。我们期望第二阶段的结果将使基于单链血管内皮生长因子的成像探针成为临床开发的可行候选。在这个项目的第二阶段，我们将采取关键步骤，在临床前开发新的靶向探针，用于特定受体的分子成像，这些受体在肿瘤血管的发生和生长中发挥关键作用，也是治疗开发的主要目标。该项目第一阶段获得的结果确立了1)使用新的专利技术构建靶向探针的可行性，以及2)获得关于这些受体状态的早期和动态信息的可行性。因此，这些靶向成像探针的临床开发为循证选择患者进行特定治疗、及时监测抗癌治疗的反应、早期癌症诊断以及区分良恶性病变打开了新的机会。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop targeted probes for imaging receptors for vascular endothelial growth factor (VEGF) in tumor angiogenesis. Overexpressed VEGF receptors play the crucial role in the onset and progression of tumor angiogenesis and therefore, these receptors are the primary targets of tremendous efforts to develop inhibitors of angiogenesis. However, these receptors are not being used as biomarkers in prescribing or monitoring anti-angiogenic therapy, because there are no non-invasive methods for assessment of their prevalence. Thus, non-invasive imaging of VEGF receptors addresses unmet clinical needs and might lead to evidence-based selection of patient for anti-angiogenic therapy, and design of personalized treatment regiments. In addition, since VEGF receptors are reportedly overexpressed in tumor and contiguous host vasculature at the onset of malignant growth, VEGF receptor imaging might be used for early diagnostics and discrimination between benign and malignant lesions. In Phase I of this project we have developed novel VEGF-based probes for near-infrared fluorescent (NIRF) and single photon emission computed tomography (SPECT) imaging of VEGF receptors that are suitable for clinical development. Site-specific conjugation of contrast agents to appropriately designed VEGF yielded probes that bind to VEGF receptors on endothelial cells as effectively as parental VEGF. We established that our VEGF-based probes undergo receptor-mediated internalization and accumulate in tumor and host endothelial cells overexpressing VEGF receptors. Importantly, using inactivated VEGF-based probes that lost the ability to bind to VEGF receptors, we evaluated a contribution of non-specific mechanisms to probe accumulation in tumor vasculature. Our targeted imaging probes are based on a novel single-chain (sc) VEGF that provides fora significant improvement in expression, refolding, and purification, relative to a conventional VEGF with two subunits linked via disulfide bonds. Site-specific conjugation of contrast agents to scVEGF via our proprietary humanized cysteine containig tag (Cys-tag) ensures homogeneity of the imaging probes. Taking together, our Phase I results open the road to clinical development of VEGF-based probes for selective and specific imaging of VEGF receptors in tumor vasculature. In Phase II, we will establish safety of scVEGF-based probes, develop scalable GLP production of conjugates and characterize utility of these probes for predicting/monitoring responses to a specific inhibitor of VEGF receptors and for early diagnostic of malignant lung lesions. We expect that Phase II results will establish scVEGF-based imaging probes as viable candidates for clinical development. In Phase II of this project we will undertake crucial steps in pre-clinical development of novel targeted probes for molecular imaging of specific receptors that play crucial role in the onset and growth of tumor vasculature and are the primary targets for therapeutic development. Results obtained in the Phase I of this project established 1) feasibility of constructing targeted probes, using a novel proprietary technology, and 2) feasibility of obtaining early and dynamic information about the status of these receptors. Thus, clinical development of these targeted imaging probes open new opportunities for evidence-based selection of patients for specific therapeutic treatments, timely monitoring responses to anti-cancer therapies, for early cancer diagnostics, and discrimination between benign and malignant lesions.