Novel High Affinity VEGF Analogs For Targeted Imaging of Thyroid Cancer

描述(申请人提供)：滤泡性甲状腺癌是最常见的内分泌组织恶性肿瘤，对女性的影响不成比例，是为数不多的发病率和患病率大幅上升的癌症之一，原因不明，而不仅仅是由于诊断的改善。大多数患者需要通过放射性碘成像进行终生诊断性监测，以发现残留和复发的肿瘤，需要随后的131I治疗。PIs之一是Genzyme的重组人促甲状腺激素(Thygen)的共同发明和共同开发，目前被批准用于增强放射性碘的诊断成像和刺激甲状腺癌血清标记物甲状腺球蛋白(TG)。然而，目前还没有最佳的方法来显示更具侵袭性的癌症数量的急剧增加，这些癌症通过血清TG升高而引起主要的发病率和死亡率，但由于Na/I同向转运体的表达减少，这些癌症已经失去了浓缩放射性碘的能力。对于这些通常高度血管生成的肿瘤，也没有任何诊断方法来预测哪些肿瘤可能对日益重要的抗血管生成治疗有反应。这两个PI发明了全新的专利方法来设计和生产亲和力更高、作用时间更长的血管内皮生长因子类似物，这些类似物可用于此类肿瘤的新型靶向成像，如广泛和令人信服的初步数据支持的那样，对间质血管内皮和肿瘤上皮中的VEGFR2受体进行靶向成像。将比较两种靶向成像方法：一种是直接优化的这种可溶性血管内皮生长因子类似物的99mTcm标记方法，另一种是使用新型聚乙二醇化和血管内皮生长因子类似物靶向“隐形”纳米颗粒的方法，该纳米颗粒可以选择性地横穿血管新生血管的高度开窗、泄漏的肿瘤血管，但不能横穿正常血管。在这一SBIR快速通道的第一阶段，PI将：(1)通过在二聚体配体两极的选定环的赖氨酸和精氨酸扫描诱变来开发更高亲和力和更长作用的hVEGF类似物，而不是通过引入新的添加两个复杂的唾液酸化碳水化合物链的新的糖基化位点；评估每个类似物与VEGFR2的结合和在刺激人血管内皮细胞(HUVEC)增殖方面的生物活性；在滚瓶中生产和纯化5-10毫克量的最终3糖化和最佳唾液酸化的类似物；(2)开发新型聚乙二醇化聚合物“隐形”纳米粒，以共价偶联的高亲和力血管内皮生长因子配体为靶点，并用偶联放射性核素标记，以获得高比活度；(3)与血管内皮生长因子类似物靶向标记纳米粒相比，评估直接偶联到99mTc或125I的血管内皮生长因子类似物在体外的结合和内化以及体内的药代动力学特性。在第二阶段，PI将：(1)在大型哺乳动物细胞生物反应器中生产和纯化最终选定的2个候选血管内皮生长因子类似物(50-200毫克)；(2)通过进一步优化成像灵敏度和特异性，极大地扩展第一阶段的体内和体外成像结果；(3)利用标记的血管内皮生长因子类似物进行平行的器官摄取定量研究；(4)最终选择特定的类似物、放射性核素和直接标记与间接纳米颗粒标记方法，基于多个标准提出商业化方案。这些全新的方法用于早期发现和定位越来越多的侵袭性甲状腺癌，应该会导致更早和更个性化的治疗，预测哪些患者将从日益重要但具有潜在毒性的抗血管生成治疗中受益最多。

DESCRIPTION (provided by applicant): Thyroid cancer of follicular origin is the most common malignancy of endocrine tissues, disproportionally affecting women, and is one of the few cancers greatly increasing in incidence & prevalence for unknown reasons not solely attributable to improved diagnosis. Most patients require lifelong diagnostic surveillance with radioiodine imaging to detect residual & recurrent tumor requiring subsequent therapy with 131I. One of the PIs co-invented & co-developed Genzyme's recombinant human TSH (Thyrogen), currently approved for enhancing diagnostic imaging with radioiodine and for stimulation of the thyroid cancer serum marker, thyroglobulin (TG). However there is currently no optimal method to image the greatly increasing number of more aggressive cancers causing major morbidity and mortality, detected by increased serum Tg, but which have lost the ability to concentrate radioiodine because of decreased expression of the Na/I symporter. Nor is there any diagnostic method to predict which tumors may respond to increasingly important anti-angiogenic therapy for these usually highly angiogenic tumors. The two PIs have invented completely novel, patented methods to design and produce much higher affinity and longer acting analogs of VEGF which can be used in novel, targeted imaging of such tumors to the VEGFR2 receptor in both the stromal vascular endothelium and in tumor epithelium, as supported by extensive and compelling preliminary data. Two approaches of targeted imaging will be compared: a direct optimized 99mTcm labeling method of such soluble VEGF analogs and another method using novel pegylated & VEGF analog targeted "stealth" nanoparticles which can selectively transverse the highly fenestrated, leaky tumor vessels of angiogenic tumor vessels but not normal vessels. In Phase 1 of this SBIR fast track the PIs will: (1) Develop higher affinity and longer acting hVEGF analogs by lysine and arginine scanning mutagenesis of selected loops in both poles of the dimeric ligand without and with introduction of a novel neoglycosylation site adding two complex, sialylated carbohydrate chains; assessing binding of each analog to VEGFR2 and bioactivity in the stimulation of human vascular endothelial cell (HUVEC) proliferation; produce and purify 5-10 mg amounts of the final 3 glycosylated and optimally sialylated analogs in CHO cells in roller bottles; (2) Develop novel pegylated polymeric "stealth" nanoparticles targeted by covalently coupled high affinity VEGF ligand and labeled with coupled radionuclides to high specific activity; (3) Assess in vitro binding and internalization of as wellas in vivo pharmacokinetic properties of VEGF analogs directly coupled to 99mTc or 125I compared to VEGF analog-targeted labeled nanoparticles. In Phase 2 the PIs will: (1) Produce and purify large amounts (50-200 mg) of the final 2 selected VEGF analog targeting candidates in a large mammalian cell bioreactor; (2) Greatly extend Phase 1 in vivo and ex vivo imaging results by further optimizing imaging sensitivity and specificity; (3) Perform parallel quantitativ organ uptake studies with labeled VEGF analogs; (4) Make the final selection of the specific analog, radionuclide and direct labeling versus indirect nanoparticle labeling method to bring forward for commercialization based on multiple criteria. These completely novel methods for earlier detection & localization of the increasing numbers of aggressive thyroid cancers should lead to earlier and more personalized therapy predicting which patients will most benefit from increasingly important but potentially toxic anti-angiogenic therapy.