Development of Superagonist Analogs of Recombinant Human TSH

描述（由申请人提供）：甲状腺癌是内分泌组织最常见的恶性肿瘤，大多数患者需要终生监测诊断性促甲状腺激素（TSH）刺激的放射性碘摄取以及放射性碘治疗的残余或转移性消融。PI与Genzyme共同发明并共同开发了目前已批准用于诊断的野生型重组人（rh） TSH，现在提议为10-20%反应不良的患者开发更有效的第二代rh TSH类似物。我们之前的结构-功能研究导致发现了TSH和其他糖蛋白激素的第一个类似物，其受体结合亲和力和生物活性显著增加（“超活性类似物”）。在目前的提案中，我们计划生产，纯化和表征大量这种新型TSH类似物用于商业开发。由于体内TSH生物测定需要比瞬时转染实验更高数量的激素，我们在目标1中建议开发稳定的细胞系，产生高水平的候选超活性类似物，并产生足够数量的初始动物研究。该目标将包括构建具有可扩增标记物和突变亚基cdna的双双性载体，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规模扩大的新型、高容量模拟净化方法。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定量和表征。在Aim中，4类似物将在现在由PI首先开发的经典小鼠rh TSH体内生物测定中进行检查，并使用完整的剂量反应曲线来评估TSH类似物的增强效力和最大功效，以便选择进一步商业开发的主要候选物。

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.