STEF as a novel antitumor agent

描述(由申请人提供)： 一种对各种不同肿瘤细胞株具有强大抗肿瘤活性的天然产物存在于短期培养的石斑鲨(Sphy Rna Tiburo)附属层细胞的培养液中。初步数据表明，该抑制因子是一种蛋白质，暂定为Sphy rna tiburo epigonal Protein-1(STEP-1)，它似乎通过阻止DNA合成和诱导增殖细胞凋亡来发挥作用。该项目的广泛和长期目标是分离活性因子，并将其提纯到可以通过重组技术生产的形式。本项目的具体目标是鉴定STEP-1的分子作用机制，并纯化具有生物活性的STEP-1。一旦纯化，将鉴定STEP-1的效力和分子作用机制，并将评估其毒性、药代动力学和体内抗肿瘤活性。生物活性STEP-1的纯化将通过采用各种生化和免疫亲和纯化策略来完成，这些策略将利用一种新的高通量流式细胞仪筛选技术，称为流式细胞术高含量筛选(FC-HCS)。还将获得纯化的STEP-1的蛋白质序列信息。用于确定STEP-1作用机制的方法包括流式细胞术、免疫印迹、Northern印迹和其他细胞凋亡和细胞周期相关蛋白的分子分析。纯化的Step-1蛋白的毒性和药代动力学将在CFU检测中使用造血祖细胞进行评估，并将在遵循标准ISO方法的一系列标准临床前毒性测试中进行测试。将在注射STEP-1的小鼠身上进行药理学研究。如果这些研究证明STEP-1具有强大的抗肿瘤活性，并具有可接受的毒性和药代动力学特性，它将为进一步进行旨在生产重组STEP-1的研究提供理由，以便在I期临床试验中进一步测试其作为抗癌剂的作用。

DESCRIPTION (provided by applicant): A natural product with potent anti-tumor activity against a variety of different tumor cell lines is present in media from short-term cultures of bonnethead shark (Sphyrna tiburo) epigonal cells. Preliminary data suggest that the inhibitory factor is a protein, tentatively termed Sphyrna tiburo epigonal protein-1 (STEP-1), and that it appears to act by blocking DNA synthesis and inducing apoptosis in proliferating cells. The broad, long-term Objectives of this project are to isolate the active factor and purify it to a form that can be produced through recombinant technology. The specific aims of the current project are to characterize the molecular mechanism of action of STEP-1 and to purify bioactive STEP-1. Once purified, the potency and molecular mechanism of action of STEP-1 will be characterized, and the toxicity, pharmacokinetics, and the in-vivo anti-tumor activity will be evaluated. Purification of bioactive STEP-1 will be accomplished by employing various biochemical and immunoaffinity purification strategies that will utilize a novel high throughput flow cytometric screening technique termed Flow Cytometric High Content Screening (FC-HCS). Protein sequence information of purified STEP-1 will also be obtained. Methods used to determine the mechanism of action of STEP-1 will include FACS, immunoblot, Northern blot and other molecular analyses of apoptosis and cell cycle associated proteins. Toxicity and pharmacokinetics of purified STEP-1 protein will be evaluated using hematopoietic progenitors in CFU assays and testing in a battery of standard preclinical toxicity assays following standard ISO methods. Pharmacologic studies will be performed in mice injected with STEP-1. If these studies demonstrate that STEP-1 has potent anti-tumor activity with acceptable toxicity and pharmacokinetic properties, it will provide the justification to proceed with future studies involving aimed at producing recombinant STEP-1 for further testing as an anti-cancer agent in phase I clinical trials.