VEGF-based Targeted Imaging of Tumor Vasculature

描述（由申请人提供）：该项目的总体目标是开发肿瘤血管生成中血管内皮生长因子（VEGF）成像受体的靶向探针。过度表达的VEGF受体在肿瘤血管生成的发生和进展中起着至关重要的作用，因此，这些受体是开发血管生成抑制剂的主要目标。然而，这些受体并没有作为生物标志物用于处方或监测抗血管生成治疗，因为没有非侵入性的方法来评估它们的患病率。因此，VEGF受体的非侵入性成像解决了未满足的临床需求，并可能导致基于证据的患者抗血管生成治疗选择，以及个性化治疗方案的设计。此外，由于VEGF受体在恶性生长开始时在肿瘤和邻近宿主血管中过表达，因此VEGF受体成像可用于早期诊断和良恶性病变的区分。在这个项目的第一阶段，我们开发了新的基于VEGF的探针，用于VEGF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，适合临床开发。造影剂与适当设计的VEGF的位点特异性偶联产生的探针与内皮细胞上的VEGF受体结合的效果与亲本VEGF一样有效。我们证实，我们的VEGF-based探针经过受体介导的内化，并在肿瘤和宿主内皮细胞中积累过表达VEGF受体。重要的是，使用失去与VEGF受体结合能力的失活VEGF探针，我们评估了非特异性机制对探测肿瘤血管积累的贡献。我们的靶向成像探针基于一种新型单链（sc） VEGF，相对于传统的具有两个亚基通过二硫键连接的VEGF，它在表达、重折叠和纯化方面有显著改善。通过我们专有的人源化含半胱氨酸标签（Cys-tag）将造影剂与scVEGF位点特异性偶联，确保成像探针的均匀性。综上所述，我们的I期研究结果为基于VEGF的探针的临床开发开辟了道路，用于肿瘤血管中VEGF受体的选择性和特异性成像。在第二阶段，我们将确定基于scvegf的探针的安全性，开发可扩展的GLP偶联物生产，并表征这些探针在预测/监测对特定VEGF受体抑制剂的反应和早期诊断恶性肺病变方面的效用。我们期望II期结果将建立基于scvegf的成像探针作为临床开发的可行候选者。在该项目的第二阶段，我们将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起着至关重要的作用，是治疗开发的主要目标。该项目的第一阶段结果确定了1)使用新颖的专有技术构建靶向探针的可行性；2)获得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发展为循证选择特定治疗方法的患者，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提供了新的机会。

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.