Development of Superactive Analogs of LH and FSH

描述（由申请人提供）：我们先前的结构-功能研究导致开发了第一种受体结合亲和力和生物活性显著增加的糖蛋白激素类似物（“超活性类似物”）。最近，我们进一步优化了人促黄体生成激素（LH）和人促卵泡激素（FSH）类似物。然而，与人促甲状腺激素（TSH）类似物相比，我们还没有研究这些类似物在体内的生物活性。这些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。 由于促性腺激素体内生物测定需要比瞬时转染实验可获得的激素量更高的激素量，我们在目的1中提出开发产生高水平候选超活性类似物的细胞系，并产生足够用于初始动物研究的量。本研究的目的是构建具有可扩增标记和突变亚基cDNA的双顺反子载体，扩增过程，生产和浓缩未纯化的类似物。在目标2中，我们建议使用目前建立的啮齿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还将包括使用四种选定类似物、两种未修饰激素和两种模拟对照进行的相应LH和FSH生物测定。这种体内生物测定的结果对于最终选择用于高水平生产和纯化的候选类似物以及随后的2期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.