Development of Superagonist Analogs of Recombinant Human TSH

重组人 TSH 超级激动剂类似物的开发

• 批准号: 7108446
• 负责人: Bruce Dale Weintraub
• 金额: $ 10.15万
• 依托单位: TROPHOGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108446
• 关键词: CHO cells; analytical ultracentrifugation; bioassay; biotechnology; dosage; drug design /synthesis /production; hormone analog; laboratory mouse; neoplasm /cancer chemotherapy; protein purification; recombinant proteins; stimulant /agonist; thyroid neoplasm; thyrotropin; transfection /expression vector

DESCRIPTION (provided by applicant): Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radioiodine uptake as well as remnant or metastatic ablation with radioidine therapy. The PI, who was both co- inventor and co-developer with Genzyme of wild-type recombinant human (rh) TSH currently approved for diagnosis, now proposes to develop more potent and more efficacious second generation analogs of rh TSH for the 10-20% of poorly responsive patients. Our previous structure- function studies resulted in the discovery of the first analogs of TSH and other glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("superactive analogs"). In the current proposal we plan to produce, purify and characterize large amounts of such novel TSH analogs for commercial development. Since TSH in vivo bioassays require higher quantities of hormones than that obtainable from transient transfection experiments, we propose in Aim 1 to develop stable cell lines producing high levels of candidate superactive analogs and produce quantities sufficient for initial animal studies. This Aim will consist of construction of dicistronic vectors with amplifiable markers and mutated subunit cDNAs, amplification, production and concentration of unpurified analogs. Aim 2 will consist of development of a novel, high capacity analog purification method suitable for commercial scale-up. Aim 3 will consist of rigorous quantification and characterization of selected purified analogs by multiple physicochemical methods, including carbohydrate analysis. In Aim 4 analogs will be examined in the now classic mouse rh TSH in vivo bioassay first developed by the PI, with full dose response curves to assess increased potency and maximal efficacy of TSH analogs in order to select a lead candidate for further commercial development.

描述(申请人提供)：甲状腺癌是最常见的内分泌组织恶性肿瘤，大多数患者需要终生监测诊断促甲状腺激素(TSH)刺激的放射性碘摄取，以及放射性核苷治疗的残余或转移消融。PI是目前被批准用于诊断的野生型重组人(Rh)TSH基因的共同发明者和共同开发者，现在建议为10%-20%的反应不良患者开发更有效和更有效的第二代rh TSH类似物。我们以前的结构-功能研究导致了TSH和其他糖蛋白激素的第一个类似物的发现，这些类似物在受体结合亲和力和生物活性方面都有很大的提高(“超活性类似物”)。在目前的提案中，我们计划生产、纯化和鉴定大量这样的新型TSH类似物，用于商业开发。由于体内TSH生物检测所需的激素水平高于瞬时转基因实验所能获得的激素水平，因此我们在目标1中建议建立稳定的细胞系，以产生高水平的候选超活性类似物，并产生足够数量的激素用于初步的动物研究。这一目标将包括构建带有可扩增标记和突变亚单位cDNA的双顺反子载体，扩增、生产和浓缩未纯化的类似物。目标2将包括开发一种适合商业放大的新的、高容量的模拟纯化方法。目标3将包括通过多种物理化学方法，包括碳水化合物分析，对选定的纯化类似物进行严格的量化和表征。在AIM中，4个类似物将在PI首次开发的经典小鼠rh TSH体内生物测定中进行检验，并用全剂量反应曲线来评估TSH类似物的增强效力和最大功效，以便为进一步的商业开发选择一个领先的候选者。