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: 8454713
• 负责人: Angelika Fath-Goodin
• 金额: $ 57.16万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454713
• 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. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of the baculovirus expression system for the production of human therapeutic proteins. This proposal addresses that deficiency and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of ParaTechs' vankyrin-enhanced baculovirus technology. Successful completion of these objectives will produce cells that provide the highest levels of accurately processed secreted and transmembrane proteins and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics.

描述（由申请人提供）：杆状病毒表达载体系统（BEV）是一种经过验证的真核蛋白表达方法。它用于生产众多研究项目的疫苗，诊断和生物活性蛋白。但是，像所有表达系统一样，BEV都有其缺点。一个事实是，由于病毒诱导的细胞死亡和裂解，表达是短暂的。 Paratechs已经商业化了一种解决此缺点的产品。表达病毒氨基蛋白酶基因的细胞系显示了杆状病毒感染细胞的延迟死亡和裂解，从而显着增强了重组蛋白的产生。这种活性被称为范吉林增强BEV（VE-BEVSTM），可将靶蛋白表达提高到22倍。杆状病毒表达的第二个局限性是，昆虫细胞缺乏产生终止溶解的，复杂的N-聚糖的能力，这限制了BEV对人类治疗蛋白表达的有用性。糖果有限责任公司已开发出一种转基因昆虫细胞系（SFSWT4），该系列表达了六种哺乳动物糖基化酶，从而允许合成终末溶蛋白的合成。该第二阶段的提案结合了Paratechs的VE技术与糖基糖细胞系，以优化“人性化” N-Glycans的表达。在第一阶段，在不同启动子的控制下，用几个范西林基因转化了转基因细胞系SFSWT4。筛选多克隆细胞以增强糖蛋白表达。数据表明，感染细胞的寿命更长，并产生更真实的溶解蛋白质证实了我们的假设。第二阶段将根据（1）根据用于生产疫苗和生物学的细胞的FDA指南来扩展VE-SWTTM细胞的克隆和表征； （2）检查VE病毒载体和VE-SWT细胞系之间的协同作用，以提供更高水平的增强； （3）证明了VE-SWTTM细胞中医学相关糖蛋白的表达增强。这些第二阶段的研究将显着扩大Paratechs VE-BEVS技术和甘露果糖的同变工程细胞系的应用。两家公司的人员都有使用要使用的技术的经验。初步研究表明，该技术有很大的机会按照设想的方式进行。此外，先前从两家公司发行的转换细胞系的先前营销经验表明，对扩展的技术有很大的需求。根据Paratechs，Inc。和Glycobac，LLC的既定声誉以及对改进的细胞系的需求，以表达重组人源化的糖蛋白的需求不断增长。 公共卫生相关性：昆虫细胞无法产生终止溶解的，复杂的N-聚糖限制了杆状病毒表达系统对生产人类治疗蛋白的有用性。该提案旨在解决缺乏症，并旨在开发新的细胞系，这些细胞系在Paratechs的Vankyrin增强杆状病毒技术的背景下产生真实的“人性化” N-聚糖。这些目标的成功完成将产生能够提供最高水平的精确分泌和跨膜蛋白的细胞，并将向从事人类分泌型和蛋白质疗法发展的结构功能研究的个别研究人员和制药公司销售。