Development of Superactive Analogs of LH and FSH

描述（由申请人提供）：我们之前的结构-功能研究导致了第一个糖蛋白激素类似物的开发，其受体结合亲和力和生物活性显著增加（“超活性类似物”）。最近，我们进一步优化了人类促黄体生成素（LH）和人类促卵泡激素（FSH）类似物。然而，与人类促甲状腺激素（TSH）类似物相比，我们尚未研究这些类似物在体内的生物活性。这种具有治疗相关终点的动物研究对于证明新开发的LH和FSH类似物的潜在治疗效用至关重要。由于体内促性腺激素生物测定需要比瞬时转染实验更高数量的激素，因此我们在目标1中建议开发能够产生高水平候选超活性类似物的细胞系，并产生足以用于初始动物研究的数量。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，扩增过程，未纯化类似物的生产和浓缩。在目标2中，我们建议使用目前建立的啮齿类动物体内生物测定法进行初步实验，以研究LH和FSH对卵母细胞产生、卵巢重量和类固醇生成的影响。Aim 2还将包括各自的LH和FSH生物测定，使用四种选定的类似物，两种未修饰的激素和两种模拟对照。这种体内生物测定的结果对于高水平生产和纯化的候选类似物的最终选择以及随后的第二阶段灵长类动物和毒理学研究至关重要。

DESCRIPTION (provided by applicant): Our previous structure-function studies resulted in the development of the first analogs of glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("super active analogs"). More recently, we have further optimized human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) analogs. However, in contrast to human thyroid-stimulating hormone (TSH) analogs, we have not yet studied bioactivity of these analogs in vivo. Such animal studies with therapeutically relevant end-points are critical to demonstrate potential therapeutic utility of newly developed LH and FSH analogs. Since the gonadotropin in vivo bioassays require higher quantities of hormones than that obtainable from transient transfection experiments we propose in Aim 1 to develop cell lines producing high levels of candidate super active 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 process, production and concentration of unpurified analogs. In Aim 2 we propose to perform initial experiments using currently established rodent in vivo bioassays modified to study the effect of LH and FSH on oocyte production, ovarian weight and steroidogenesis. The Aim 2 will also include respective LH and FSH bioassays with four selected analogs, two unmodified hormones and two mock controls. The results of such in vivo bioassays will be essential in the final selection of candidate analogs for high-level production and purification, as well as subsequent phase 2 primate and toxicology studies.