Enhancing mammalian glycoprotein production in the baculovirus expression vector

增强杆状病毒表达载体中哺乳动物糖蛋白的产量

• 批准号: 8550085
• 负责人: Angelika Fath-Goodin
• 金额: $ 49.71万
• 依托单位: PARATECHS CORP.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8550085
• 关键词: Address; Alteplase; Ankyrins; Baculovirus Expression System; Baculoviruses; Biological; Cell Death; Cell Line; Cells; Cessation of life; Cloning; Complex; Cytolysis; Data; Development; Diagnostic; Disadvantaged; Engineering; Enzymes; Funding; Genes; Glycoproteins; Goals; Guidelines; Human; Human Resources; Individual; Influenza Virus Hemagglutinin Glycoproteins; Insecta; Integral Membrane Protein; Life; Marketing; Methods; Modeling; Pharmacologic Substance; Phase; Polysaccharides; Positioning Attribute; Process; Production; Property; Proteins; Recombinant Proteins; Recombinants; Research Personnel; Research Project Grants; Serum-Free Culture Media; Site; Structure; System; Techniques; Technology; Testing; Transformed Cell Line; Transgenic Organisms; United States National Institutes of Health; Vaccines; Viral; Virus; alpha 1-Antitrypsin; base; efficacy testing; experience; expression vector; glycosylation; improved; new technology; phase 2 study; promoter; protein expression; therapeutic protein; vector

DESCRIPTION (provided by applicant): The baculovirus expression vector system (BEVS) is a proven, powerful and versatile method of eukaryotic protein expression. It is used to produce vaccines, diagnostics, and biologically active proteins for a multitude of research projects. Like all expression systems, however, BEVS has its disadvantages. One is the fact that expression is short-lived due to virus-induced cell death and lysis. ParaTechs has already commercialized a product that addresses this shortcoming. Cell lines that express a viral ankyrin gene show delayed death and lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production. This activity, referred to as vankyrin-enhanced BEVS (VE-BEVSTM), boosts target protein expression up to 22-fold. A second limitation of baculovirus expression is that insect cells lack the ability to produce terminally sialylated, complex N-glycans, which limits the usefulness of BEVS for the expression of human therapeutic proteins. GlycoBac LLC has developed a transgenic insect cell line (SfSWT4) that expresses six mammalian glycosylation enzymes, allowing synthesis of terminally sialyated proteins. This Phase II proposal combines ParaTechs' VE technology with GlycoBac's cell line to optimize expression of "humanized" N-glycans. In Phase I, the transgenic cell line SfSWT4 was transformed with several vankyrin genes under the control of different promoters. Polyclonal cells were screened for enhanced glycoprotein expression. Data demonstrating that infected cells lived longer and produced more authentically sialylated protein confirmed our hypothesis. Phase II will extend these studies by (1) cloning and characterization of VE-SWTTM cells according to FDA guidelines for cells used to produce vaccines and biological; (2) examining synergistic effects between VE virus vectors and VE-SWT cell lines to provide greater levels of enhancement; and (3) demonstrating enhanced expression of medically relevant glycoproteins in VE-SWTTM cells. These Phase II studies will significantly expand the applications of ParaTechs' VE-BEVS technology and GlycoBac's glycoengineered cell lines. Personnel at both companies have experience with the techniques to be used. Preliminary studies indicate that this technology has a significant chance of performing as envisioned. Furthermore, prior marketing experience with transformed cell lines previously released from the two companies suggests a significant demand for the expanded technology. This new technology should be relatively easy to commercialize based on the established reputations of ParaTechs, Inc. and GlycoBac, LLC and the growing demand for improved cell lines to express recombinant humanized glycoproteins.

描述（由申请人提供）：杆状病毒表达载体系统（BEVS）是一种经证实的、功能强大且通用的真核蛋白表达方法。它被用于生产疫苗，诊断和生物活性蛋白质，用于许多研究项目。然而，与所有表达系统一样，BEVS也有其缺点。一个是由于病毒诱导的细胞死亡和裂解，表达是短暂的。ParaTechs已经将一种解决这一缺陷的产品商业化。表达病毒锚蛋白基因的细胞系显示杆状病毒感染细胞的延迟死亡和裂解，从而显著增强重组蛋白的产生。这种活性被称为vanklavin增强的BEVS（VE-BEVSTM），可将靶蛋白表达提高22倍。杆状病毒表达的第二个限制是昆虫细胞缺乏产生末端唾液酸化的复合N-聚糖的能力，这限制了BEVS用于表达人治疗性蛋白质的有用性。GlycoBac LLC开发了一种转基因昆虫细胞系（SfSWT 4），该细胞系表达六种哺乳动物糖基化酶，允许合成末端唾液酸化蛋白。该II期提案将ParaTechs的VE技术与GlycoBac的细胞系相结合，以优化“人源化”N-聚糖的表达。在第一阶段，转基因细胞系SfSWT 4用几个vankyrin基因在不同的启动子的控制下转化。筛选多克隆细胞以增强糖蛋白表达。数据表明，受感染的细胞寿命更长，产生更多的真正唾液酸化的蛋白质证实了我们的假设。II期将通过以下方式扩展这些研究：（1）根据FDA关于用于生产疫苗和生物制品的细胞的指导原则克隆和表征VE-SWTTM细胞;（2）检查VE病毒载体和VE-SWT细胞系之间的协同效应，以提供更高水平的增强;以及（3）证明VE-SWTTM细胞中医学相关糖蛋白的表达增强。这些II期研究将显著扩大ParaTechs的VE-BEVS技术和GlycoBac的糖工程细胞系的应用。两家公司的人员都有使用技术的经验。初步研究表明，这项技术有很大的机会按照设想执行。此外，先前从这两家公司发布的转化细胞系的营销经验表明，对扩展技术的需求很大。根据ParaTechs，Inc.的既定声誉，这项新技术应该相对容易商业化。和GlycoBac，LLC，以及对表达重组人源化糖蛋白的改良细胞系的日益增长的需求。

项目成果

Improving the baculovirus expression vector system with vankyrin-enhanced technology.

通过Vankyrin增强技术改善杆状病毒表达载体系统。

• DOI: 10.1002/btpr.2516
• 发表时间: 2017-11
• 期刊: Biotechnology progress
• 影响因子: 2.9
• 作者: Steele KH;Stone BJ;Franklin KM;Fath-Goodin A;Zhang X;Jiang H;Webb BA;Geisler C
• 通讯作者: Geisler C