Trimer-Tag: A Technology for Producing Trivalent Biologics

描述（由申请人提供）：治疗自身免疫性疾病如类风湿性关节炎和银屑病的现代策略之一涉及使用生物TNF受体诱饵，如可溶性受体或治疗性抗体，以阻断炎性配体TNF-1，从而阻断其受体的病理活化。然而，目前的TNF-1生物阻断剂在结构上都是二聚体，而TNF-1本身在性质上是同源三聚体。从结构生物学的角度来看，具有双重对称性的同源二聚体结构不能完美地对接到具有三重对称性的同源三聚体结构，从而限制了两个分子之间的亲和力。在这里，我们描述了一个通用的方法，有效地创建三聚体可溶性受体作为分泌蛋白。该过程涉及具有配体结合结构域或任何生物活性蛋白的可溶性受体与来自前胶原的C-前肽结构域的三聚化标签（三聚体标签）之间的基因融合，所述三聚化标签能够自组装成二硫键连接的三聚体。我们表明，同源三聚体的可溶性TNF受体与这种方法产生的是一个更有效的阻断剂比二聚体TNF受体诱饵抑制TNF- 1信号在体外。此外，我们还证明了共价增强的同源三聚体TRAIL/Apo 2L-三聚体配体是一种有效的抗癌剂，与其二聚体Fc融合对应物相反。因此，Trimer-Tag有可能成为合理设计下一代生物药物的新平台技术，用于治疗自身免疫性疾病，癌症，艾滋病，骨质疏松症和心脏病。在这项I期SBIR应用中，我们寻求显着提高这些重组三聚体融合蛋白的表达水平并优化其纯化方案，希望这项由3项美国专利覆盖的新技术能够迅速从临床前阶段走向数百万患者的床边。 公共卫生相关性：这一阶段SBIR申请旨在进一步优化和简化新专利的蛋白质三聚化技术，用于设计和生产针对自身免疫性疾病、癌症、艾滋病、骨质疏松症和心脏病等主要疾病的分泌型治疗性生物制剂。

DESCRIPTION (provided by applicant): One of the modern strategies for treating autoimmune diseases such as rheumatoid arthritis and psoriasis involves the use of biologic TNF receptor decoys, such as soluble receptors or therapeutic antibodies, to intercept the inflammatory ligand TNF-1, and thus block the pathological activation of its receptors. However, current TNF-1 biologic blockers are all dimeric in structure, whereas TNF-1 itself is homotrimeric in nature. From a structural biology point of view, a homodimeric structure with a two-fold symmetry cannot perfectly dock to a homotrimeric structure with a three-fold symmetry, thus limiting the affinity between the two molecules. Here we describe a general methodology for efficient creation of trimeric soluble receptors as secreted proteins. The process involves gene fusion between a soluble receptor with a ligand binding domain or any biologically active protein and a trimerization tag from the C-propeptide domain of pro-collagen (Trimer-Tag), which is capable of self-assembly into a disulfide bond-linked trimer. We show that the homotrimeric soluble TNF receptor produced with such method is a more potent blocker than dimeric TNF receptor decoys in inhibiting TNF- 1 signaling in vitro. Moreover, we have also demonstrated that covalently strengthened homotrimeric TRAIL/Apo2L-Trimer ligand is a potent anticancer agent, in contrast to its dimeric Fc fusion counterpart. Thus, Trimer-Tag has the potential to become a new platform technology for rational design of the next generation biologic drugs against autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease. In this Phase I SBIR application, we seek to significantly increase the expression level and optimize the purification scheme of these recombinant trimeric fusion proteins in the hope that this novel technology, which is covered by 3 U.S. patents, can quickly move from preclinical stage towards the bedsides of millions of patients. PUBLIC HEALTH RELEVANCE: This Phase I SBIR application seeks to further optimize and streamline a newly patented protein trimerization technology for the design and production of secreted therapeutic biologics targeting major diseases such as autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease.