Trimer-Tag: A Technology for Producing Trivalent Biologics

Trimer-Tag：一种生产三价生物制剂的技术

• 批准号: 8200347
• 负责人: PENG LIANG
• 金额: $ 22.47万
• 依托单位: GENHUNTER CORPORATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8200347
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Antineoplastic Agents; Arthritis; Aspartate; Autoimmune Diseases; Bacteria; Bacteriophages; Bioreactors; Carbamoyl Transferases; Cells; Chimeric Proteins; Chinese Hamster Ovary Cell; Chromatography; Clinical; Codon Nucleotides; Collagen; Collagen Fibril; Complex; Disease; Docking; Drug Design; Escherichia; Etanercept; Event; Extracellular Matrix; Family; Frequencies; Gene Amplification; Gene Fusion; Genetic Transcription; Glycine; HIV; HIV Envelope Protein gp120; Heart Diseases; Homo; Human; Human body; In Vitro; Infection; Inflammatory; Injection of therapeutic agent; Intercept; Legal patent; Ligand Binding Domain; Ligands; Link; Logistics; Malignant Neoplasms; Mammals; Marketing; Mediating; Methodology; Methods; Molecular; Monoclonal Antibodies; N-terminal; Nature; Non-Human Protein; Osteoporosis; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Play; Process; Procollagen; Production; Proteins; Psoriasis; Recombinants; Rheumatoid Arthritis; Scheme; Serum; Signal Transduction; Site; Small Business Innovation Research Grant; Staging; Structure; Subcutaneous Injections; Surface Antigens; Suspension Culture; TNF gene; TNFSF10 gene; TNFSF11 gene; Technology; Therapeutic; Therapeutic antibodies; Tissues; Translations; Tumor Necrosis Factor Receptor; Viral; Yeasts; analog; base; bioprocess; cost; cytokine; design; disulfide bond; expression cloning; fibrous protein; gene synthesis; immunogenicity; member; molecular assembly/self assembly; new technology; next generation; polypeptide; pre-clinical; procollagen C-endopeptidase; receptor; response; self assembly; structural biology; therapeutic target; triple helix

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.

描述(申请人提供)：治疗类风湿性关节炎和牛皮癣等自身免疫性疾病的现代策略之一涉及使用生物肿瘤坏死因子受体诱饵，如可溶性受体或治疗性抗体，以拦截炎症配体肿瘤坏死因子-1，从而阻止其受体的病理激活。然而，目前的肿瘤坏死因子-1生物阻滞剂在结构上都是二聚体，而肿瘤坏死因子-1本身在性质上是同源三聚体。从结构生物学的角度来看，具有两重对称性的同源二聚体结构不能与具有三重对称性的同源三聚体结构完美对接，从而限制了两个分子之间的亲和力。在这里，我们描述了一种有效地创建三聚体可溶性受体作为分泌蛋白的一般方法。这一过程涉及到带有配体结合域的可溶性受体或任何生物活性蛋白与前胶原C-前肽结构域的三聚化标签(Trimer-Tag)之间的基因融合，三聚体标签能够自组装成二硫键连接的三聚体。我们证明，用这种方法生产的高三聚体可溶性肿瘤坏死因子受体在体外抑制肿瘤坏死因子-1信号转导方面比二聚体可溶性肿瘤坏死因子受体诱饵更有效。此外，我们还证明了共价强化的高三聚体TRAIL/Apo2L-三聚体配体是一种有效的抗癌药物，而不是它的二聚体FC融合配体。因此，Trimer-Tag有可能成为合理设计治疗自身免疫性疾病、癌症、艾滋病、骨质疏松症和心脏病的下一代生物药物的新平台技术。在这项第一阶段的SBIR应用中，我们寻求显著提高这些重组三聚体融合蛋白的表达水平并优化纯化方案，希望这项拥有3项美国专利的新技术能够迅速从临床前阶段进入数百万患者的床边。 公共卫生相关性：这项第一阶段的SBIR应用旨在进一步优化和简化一项新获得专利的蛋白质三聚技术，以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重大疾病的分泌型治疗性生物制剂。