Enhancing mammalian glycoprotein production in the baculovirus expression vector

描述(申请人提供)：杆状病毒表达载体系统(BEVS)是一种成熟、强大和通用的真核蛋白表达方法。它被用来为许多研究项目生产疫苗、诊断和生物活性蛋白。然而，像所有的表达系统一样，BEVS也有它的缺点。其一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Ankyrin基因的细胞系表现出杆状病毒感染细胞的延迟死亡和裂解，从而显著提高重组蛋白的产量。这种被称为vankyrin增强型BEVS(VE-BEVSTM)的活动，将目标蛋白的表达提高了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杂N-聚糖的能力，这限制了BEVS在表达人类治疗性蛋白方面的有效性。GlycoBac LLC开发了一种转基因昆虫细胞系(SfSWT4)，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。这一第二阶段的方案结合了ParaTechs的VE技术和GlycoBac的细胞系，以优化“人源化”N-糖链的表达。在第一阶段，将多个vankyrin基因在不同启动子的控制下转化到转基因细胞系SfSWT4中。对多克隆细胞进行糖蛋白表达增强筛选。数据显示，受感染的细胞寿命更长，产生更多真正的唾液酸化蛋白，这证实了我们的假设。第二阶段将通过(1)根据FDA用于生产疫苗和生物的细胞指南克隆和鉴定VE-SWTTM细胞；(2)检验VE病毒载体和VE-SWT细胞系之间的协同效应以提供更大程度的增强；以及(3)展示VE-SWTTM细胞中医学相关糖蛋白的增强表达，从而扩展这些研究。这些二期研究将大大扩展ParaTechs的VE-BEVS技术和GlycoBac的糖工程细胞系的应用。这两家公司的人员都对将要使用的技术有经验。初步研究表明，这项技术很有可能实现预期的效果。此外，这两家公司之前发布的转化细胞系的先前营销经验表明，对扩展技术的需求很大。基于ParaTechs，Inc.和GlycoBac，LLC确立的声誉，以及对改进细胞系以表达重组人源化糖蛋白的日益增长的需求，这项新技术应该相对容易商业化。

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.