Development of Superagonist Analogs of Recombinant Human TSH for Imaging and Ther

描述（由申请人提供）：甲状腺癌越来越普遍，是内分泌组织中最常见的恶性肿瘤，大多数患者需要终身监测诊断促甲状腺激素（TSH）刺激的放射性碘摄取以及甲状腺残留或肿瘤消融放射性碘治疗。该PI与Genzyme共同发明并共同开发了野生型重组人（rh） TSH（甲状腺激素），目前已被批准用于刺激诊断性放射性碘摄取和甲状腺球蛋白分泌，现在提议开发一种更有效的第二代rh TSH过度活性类似物，用于诊断成像中15-20%反应不良的甲状腺癌患者以及大多数此类患者的放射性碘治疗。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。在我们获得高分并成功完成的1期SBIR研究中，我们实现了所有目标，包括开发稳定的细胞系，产生高水平的最终两种候选超活性类似物；大型生物反应器生产方法的优化开发一种适合商业规模的新型、高容量净化方法；通过多种物理化学方法对纯化类似物进行严格的定量和表征，并证明了现在由PI首先开发的经典体内小鼠rh TSH体内生物测定法的效力和功效。在目前的二期计划中，我们已经提供了广泛的初步数据，远远超出了一期计划的目标，我们计划在纤维床生物反应器中生产和纯化最后两种TSH类似物候选TR1401和1402的额外量（bbb200毫克），足以用于所有二期体内研究，使用在一期研究中优化的生产和纯化方法。我们还计划开发和验证一种新的免疫测定方法，用于检测未纯化的啮齿动物血浆中的TSH类似物，该方法可以准确地定量，并与严格的HPLC方法相同，然后用于确定啮齿动物的IV代谢清除率和IP， SC和IM药代动力学。最重要的是，在这个第二阶段的提案中，我们将首次在正常小鼠、Ret/PTC1和其他甲状腺癌转基因小鼠模型中，比较TSH类似物TR1401和1402与野生型（Genzyme）重组TSH作用的多个临床相关诊断和治疗终点。具体来说，我们将通过直接器官计数、124I和18氟脱氧葡萄糖诊断性甲状腺PET成像以及治疗性131I诱导的肿瘤消退来检查野生型和模拟TSH在甲状腺125I摄取中的详细剂量反应曲线。最后，我们将确定TR1401和1402与选定的新开发的蛋白激酶抑制剂在体外和体内的诊断和治疗作用的可能互补。公共卫生相关性：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促甲状腺激素（TSH）刺激放射碘成像诊断以发现复发肿瘤。PI和共同研究者已经开发了一种改进的TSH形式，并提出了一种全新的甲状腺癌成像方法，这将大大提高早期发现，并改善癌症治疗和生存率。

DESCRIPTION (provided by applicant): Thyroid cancer is increasingly prevalent and 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 thyroid remnant or tumor ablation with radioiodine therapy. The PI, who was both co-inventor and co-developer with Genzyme of wild type recombinant human (rh) TSH (Thyrogen) currently approved for stimulation of diagnostic radioiodine uptake and thyroglobulin secretion, now proposes to develop a more potent and more efficacious second generation superactive analog of rh TSH for the 15-20% of poorly responsive thyroid cancer patients in diagnostic imaging as well as for the majority of such patients for radioiodine therapy. 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. During our highly scored and successfully completed phase 1 SBIR study we have achieved all the aims including developing stable cell lines producing high levels of the final two candidate superactive analogs; optimization of large scale bioreactor production methods; development of a novel, high capacity purification methods suitable for commercial scale-up; rigorous quantification and characterization of purified analogs by multiple physicochemical methods, and proof of increased potency and efficacy in the now classic in vivo mouse rh TSH in vivo bioassay first developed by the PI. In the current phase 2 proposal, for which we have provided extensive preliminary data well beyond the aims of the phase 1 proposal, we plan to produce and purify additional large amounts (>200mg) of the final two TSH analog candidates TR1401 and 1402 in the fibrous bed bioreactor sufficient for all the phase two in vivo studies using the methods of production and purification optimized in the phase one study. We also plan to develop and validate a novel immunoassay for the detection of TSH analogs in unpurified rodent plasma that quantifies accurately and identically to rigorous HPLC methods which will then be used to determine the IV metabolic clearance rate and the IP, SC and IM phamacokinetics in rodents. Most importantly, in this phase two proposal we will examine for the first time multiple clinically relevant diagnostic and therapeutic endpoints of TSH action comparing TSH analogs TR1401 and 1402 to wild type (Genzyme) recombinant TSH in normal mice and in those with Ret/PTC1 and other transgenic mouse models of thyroid cancer. Specifically we will examine detailed dose response curves for both wild type and analog TSH in thyroidal 125I uptake by direct organ counting, diagnostic thyroidal PET imaging with 124I and 18 Fluorodeoxyglucose as well as therapeutic 131I -induced tumor regression. Finally, we will determine the possible complementation of the diagnostic and therapeutic effects of TR1401 and 1402 with those of selected newly developed protein kinase inhibitors in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radio-iodine imaging to detect recurrent tumor. The PI and Co- Investigator have developed a much improved form of TSH and propose a completely new method of thyroid cancer imaging that should greatly improve early detection and lead to improved cancer treatment and survival.