Use of a viral mucin-like protein to convert adherent cells to suspension culture

描述(申请人提供)：大多数细胞培养系依赖于贴壁，需要表面贴附才能增殖。对于工业生产，微载体微珠可以提供更大的表面积，但相关的成本增加和由此产生的操作复杂性可能会阻止它们的使用。由于这些限制，不依赖于锚定的细胞是生产生物药物的首选细胞，但适当的不依赖于锚定的细胞并不适用于所有应用；例如，生产疫苗和细胞类型特定蛋白。这项提案探索了使用一种新的昆虫病毒蛋白将贴壁细胞转化为能够在悬浮培养中茁壮成长的细胞。ParaTechs已经从Agrotis ipsilon(黑地老虎)中发现了一种细胞系，它提供了比标准Sf9细胞系高3到10倍的重组蛋白表达水平。不幸的是，这些细胞具有很强的粘附性，这使得它们不适合大规模生产蛋白质。我们打算将一种昆虫病毒基因稳定地转化到A.ipsilon细胞中，这种基因会导致血细胞的粘附性丧失。我们预测，这种蛋白在转化细胞中的表达将使它们能够在悬浮培养中生长。与我们的Vankyrin增强型杆状病毒表达载体系统(VE-BEVS)相结合，我们预计每个细胞的蛋白质表达水平将至少比目前可能的水平高12至220倍。产量的这一戏剧性增长将对所有BEVS用户，从个人研究人员到大型生物制药公司都具有重大意义。我们还将把这项技术应用于贴壁哺乳动物组织培养。一种将哺乳动物细胞从非锚定培养转化为悬浮培养的简单方法将对疫苗和生物疗法的生产做出重大贡献，并将改善人类健康。ParaTechs的工作人员是经验丰富的杆状病毒学家和细胞生物学家。这些协议使用我们公司经常采用的标准技术。我们预计试验不会有困难。目前的文献有力地支持了我们的假设，我们对我们的成功充满信心。 公共卫生相关性：人类医学的未来将涉及以蛋白质为基础的药物和亚单位疫苗的开发，其生产将需要在悬浮培养中大规模繁殖组织培养细胞。然而，大多数连续的细胞系依赖于锚定，并且没有方法可以常规和容易地将贴壁细胞转化为悬浮培养。ParaTechs将测试一种导致血细胞失去粘附性的昆虫病毒基因，以使依赖锚定的昆虫和哺乳动物细胞适应悬浮培养。这一目标的实现将对生物药剂学和人类健康做出重大贡献。

DESCRIPTION (provided by applicant): Most cell culture lines are anchorage-dependent and require surface attachment for proliferation. For industrial production, increased surface area can be provided by microcarrier beads, but the associated increases in cost and the resulting complexity of manipulation may preclude their use. Due to these limitations, anchorage-independent cells are preferred for the production of biopharmaceuticals, but appropriate anchorage-independent cells are not available for all applications; e.g., production of vaccines and cell-type specific proteins. This proposal explores the use of a novel insect virus protein to transform adherent cells to cells that can thrive in suspension culture. ParaTechs has identified a cell line from Agrotis ipsilon (black cutworm), which provides levels of recombinant protein expression that are 3-to-10 times higher than the standard Sf9 cell line. Unfortunately, the cells are strongly adherent, which makes them unsuitable for large-scale protein production. We intend to stably transform the A. ipsilon cells with an insect virus gene that causes a loss of adhesion in hemocytes. We predict that expression of this protein in transformed cells will enable them to grow in suspension culture. In combination with our Vankyrin-Enhanced Baculovirus Expression Vector System (VE-BEVS), which has the ability to increase protein production per cell by a factor of 4 to 22, we anticipate achieving a level of protein expression per cell that is at least 12-to-220 times higher than currently possible. This dramatic increase in yield will be significant for all BEVS users, from individual researchers to large biopharmaceutical companies. We also will apply this technology to adherent mammalian tissue cultures. A simple method for converting mammalian cells from anchorage-independent to suspension culture would make a significant contribution to the production of vaccines and bio-therapeutics, and would result in improved human health. ParaTechs personnel are experienced baculovirologists and cell biologists. The protocols use standard technologies that are routinely adopted in our company. We do not anticipate difficulty with the experimentation. Current literature strongly supports our hypothesis and we are confident of our success. PUBLIC HEALTH RELEVANCE: The future of human medicine will involve a dramatic increase in development of protein-based drugs and subunit vaccines, whose production will require large-scale propagation of tissue culture cells in suspension culture. Most continuous cell lines, however, are anchorage-dependent, and no method exists to routinely and easily transform adherent cells to suspension culture. ParaTechs will test an insect virus gene that causes hemocytes to lose adhesion for the ability to adapt anchorage- dependent insect and mammalian cells to suspension culture. Achievement of this goal would make a significant contribution to biopharmacology and human health.