Novel High Affinity VEGF Analogs For Targeted Imaging of Thyroid Cancer

描述（由申请人提供）：滤泡源性甲状腺癌是内分泌组织中最常见的恶性肿瘤，对女性的影响尤为严重，是为数不多的发病率和患病率大幅增加的癌症之一，原因不明，并非完全归因于诊断的改善。大多数患者需要终身使用放射性碘显像进行诊断监测，以发现残余和复发肿瘤，并需要后续的131I治疗。其中一个pi共同发明并共同开发了重组人TSH（甲状腺激素），目前已被批准用于增强放射性碘的诊断成像和甲状腺癌血清标志物甲状腺球蛋白（TG）的刺激。然而，目前还没有最佳的方法来成像数量急剧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，通过血清Tg的增加来检测，但由于Na/I同调体的表达减少，这些癌症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的日益重要的抗血管生成治疗有反应。这两家pi已经发明了完全新颖的专利方法来设计和生产高亲和力和长效的VEGF类似物，这些类似物可以用于这种肿瘤的新型靶向成像，在基质血管内皮和肿瘤上皮中都有VEGFR2受体，这得到了广泛和令人信服的初步数据的支持。我们将比较两种靶向成像方法：一种是直接优化这种可溶性VEGF类似物的99mTcm标记方法，另一种是使用新型聚乙二醇化和VEGF类似物靶向“隐形”纳米颗粒的方法，这种纳米颗粒可以选择性地穿过血管生成肿瘤血管的高度开窗、渗漏的肿瘤血管，而不是正常血管。在SBIR快速通道的第一阶段，pi将：(1)通过赖氨酸和精氨酸扫描诱变，在二聚体配体的两端选择环，而不引入新的糖基化位点，添加两个复杂的唾液化碳水化合物链，从而开发出高亲和力和长效的hVEGF类似物；评估每种类似物与VEGFR2的结合以及在刺激人血管内皮细胞（HUVEC）增殖中的生物活性；在滚轮瓶中的CHO细胞中产生和纯化5-10毫克量的最终3糖基化和最佳唾液化类似物；(2)开发新型聚乙二醇化聚合物“隐身”纳米粒子，以共价偶联的高亲和力VEGF配体为靶标，并用偶联的放射性核素标记高比活性；(3)与VEGF类似物靶向标记纳米颗粒相比，评估直接偶联于99mTc或125I的VEGF类似物的体外结合、内化和体内药代动力学特性。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种VEGF类似物（50-200 mg）；(2)通过进一步优化成像灵敏度和特异性，大大延长了一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)根据多种标准，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，提出商业化方案。这些全新的方法用于越来越多的侵袭性甲状腺癌的早期检测和定位，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具有潜在毒性的抗血管生成治疗中获益。

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.