Novel High Affinity VEGF Analogs For Targeted Imaging of Thyroid Cancer

描述（由申请人提供）：滤泡性甲状腺癌是内分泌组织最常见的恶性肿瘤，严重影响女性，是少数几种发病率和患病率大幅增加的癌症之一，原因不明，不仅仅是由于诊断的改善。大多数患者需要放射性碘成像进行终身诊断监测，以检测需要后续131 I治疗的残留和复发肿瘤。其中一名PI共同发明和共同开发了Genzyme的重组人TSH（Thyrogen），目前已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志物甲状腺球蛋白（TG）。然而，目前没有最佳的方法来成像数量大大增加的更具侵袭性的癌症，这些癌症导致主要的发病率和死亡率，通过增加的血清Tg检测，但是由于Na/I同向转运体的表达降低而失去了浓缩放射性碘的能力。也没有任何诊断方法来预测哪些肿瘤可能对这些通常高度血管生成的肿瘤越来越重要的抗血管生成治疗有反应。这两名PI发明了全新的专利方法，用于设计和生产亲和力更高、作用时间更长的VEGF类似物，这些类似物可用于此类肿瘤对基质血管内皮和肿瘤上皮中VEGFR 2受体的新型靶向成像，这得到了广泛和令人信服的初步数据的支持。将比较两种靶向成像的方法：这种可溶性VEGF类似物的直接优化的99 mTcm标记方法和使用新型聚乙二醇化和VEGF类似物靶向“隐形”纳米颗粒的另一种方法，所述纳米颗粒可以选择性地穿过血管生成肿瘤血管的高度有孔的、渗漏的肿瘤血管，而不是正常血管。（1）通过赖氨酸和精氨酸扫描诱变二聚体配体两极中的选定环，在不引入和引入新的新糖基化位点的情况下，添加两个复杂的唾液酸化糖链，开发更高亲和力和更长效的hVEGF类似物;评估每种类似物与VEGFR 2的结合和在刺激人血管内皮细胞（HUVEC）增殖中的生物活性;在转瓶中的CHO细胞中产生并纯化5-10 mg量的最终3种糖基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”纳米颗粒，其被共价偶联的高亲和力VEGF配体靶向并用偶联的放射性核素标记至高比活性;（3）与VEGF类似物靶向标记的纳米颗粒相比，评估直接偶联至99 mTc或125 I的VEGF类似物的体外结合和内化以及体内药代动力学性质。在第二阶段，PI将：（1）产生和纯化大量（50-200 mg）的最终2种选择的VEGF类似物靶向候选物;（2）通过进一步优化成像灵敏度和特异性，极大地延长了1期体内和离体成像结果;（3）用标记的VEGF类似物进行平行定量器官摄取研究;（4）根据多项标准，最终选择特异性类似物、放射性核素和直接标记与间接纳米颗粒标记方法，提出商业化。这些用于早期检测和定位越来越多的侵袭性甲状腺癌的全新方法应该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潜在毒性的抗血管生成治疗中获益最多。

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.