Investigation and manipulation of mTOR cellular signalling to generate novel CHO host cells with high growth and productivity characteristics

小分子药物（例如抗生素）传统上是人类治疗和疗法的支柱，然而在过去的10-20年中，基于蛋白质的药物（例如赫赛汀，通常用于治疗乳腺癌）已经发展，使得这些药物现在构成了医药市场的重要部分。有几类基于蛋白质的药物，其中之一，单克隆抗体，在使用或临床试验中构成一类中最大数量的蛋白质分子。许多基于蛋白质的药物的生产具有挑战性，因为它们（a）需要特定的辅助蛋白来折叠和组装成它们的最终活性状态，并且（B）在它们的表面上被糖和对其生物活性至关重要的其他分子修饰。由于生产这种生物治疗剂所需的高精度，这种用于治疗疾病的基于“重组”蛋白质的药物通常由在限定条件下培养的细胞生产。其中一个问题是，我们用来制造蛋白质用于治疗用途的细胞并不像我们希望的那样有效。因此，我们可能无法生产足够的这些药物和/或生产它们的成本可能太高，卫生保健提供者。因此，该提案旨在解决支持从哺乳动物细胞合成重组蛋白的关键领域。它的目的是提供蛋白质合成的全球调节因子mTOR（mammalian target of rapamycin）如何有助于重组蛋白质合成的理解，并设计新的方法来操纵这一过程，以提高重组蛋白质产量。蛋白质合成是细胞中遗传物质中的信息通过中间信使（称为mRNA）转化为蛋白质（如抗体）的过程。蛋白质合成参与核糖体，并且mTOR信号传导也影响核糖体生物合成，因此是研究生物蛋白质合成的关键靶标。当这些细胞在培养物中生长时产生的重组蛋白的量由可以产生的细胞的数量（“生物量”）和由每个细胞产生的重组生物素蛋白的量（所谓的“细胞比生产率”）决定。雷帕霉素（或mTOR）的哺乳动物靶标是细胞生长和增殖（因此是生物量）以及蛋白质合成的主要调节剂。然而，这仍然是建立如何主调节有助于重组蛋白的合成，以及是否可以操纵mTOR，以提高生长和产品水平。因此，令人惊讶的是，还没有关于mTOR信号传导在确定哺乳动物细胞的重组蛋白产量中可能发挥的作用的全面研究。我们将进行这样的研究，调查我们的总体假设（i）哺乳动物重组细胞系的生长和生产力是由mTOR（细胞增殖、核糖体生物合成和蛋白质合成的全局调节因子）支撑的，以及（ii）这种全局调节因子和信号传导途径的工程化增加了细胞分裂的速率，并增强了CHO细胞的重组蛋白生产。总体目标是产生新的哺乳动物细胞，其利用mTOR信号传导的操纵来增强单克隆抗体和其他重组产物的产生。该信息与工业非常重要相关，因为当细胞在培养物中稍后变得应激时以及生物反应器中积累的生物质的量阻碍了商业上有价值的蛋白质（例如抗体）的生产。如果没有改进的表达系统，生物技术/制药工业将缺乏生产足够大量的这些有价值和有效的药物以满足需求的能力，所述药物的价格将允许将它们开给将从中受益的患者。

Small molecule drugs (e.g. antibiotics) have traditionally been the mainstay of treatments and therapies in man, however in the last 10-20 years protein based drugs (e.g. herceptin, often used to treat breast cancer) have developed such that these now constitute a significant section of the pharmaceutical market. There are several categories of protein based drugs, one of which, monoclonal antibodies, constitutes the largest number of protein molecules in a class either in use or in clinical trials. Many protein based drugs are challenging to produce because they (a) require particular helper proteins to fold and assemble into their final active state and (b) are decorated on their surfaces by sugars and other molecules that are essential to their bioactivity. Due to the high precision required to produce such biotherapeutics, such 'recombinant' protein-based drugs for the treatment of diseases are usually produced by cells kept in culture under defined conditions. One problem with this is that the cells we use to make proteins for therapeutic uses are not as efficient as we would like them to be. As a consequence, we may not be able to produce enough of these drugs and/or the cost of producing them may be too high for health care providers. This proposal therefore sets out to address a key area that underpins recombinant protein synthesis from mammalian cells. It aims to provide understanding of how a global regulator of protein synthesis, mTOR (mammalian target of rapamycin), contributes to recombinant protein synthesis and devise new ways to manipulate this process to enhance recombinant protein yields.Protein synthesis is the process by which the information in the genetic material in the cell, DNA is converted via an intermediary messenger, termed mRNA, into proteins such as antibodies. Protein synthesis takes part on ribosomes and mTOR signalling also effects ribosomes biogenesis and hence is a key target to investigate with respect to biotherapeutic protein synthesis. The amount of recombinant protein produced when these cells are grown in culture is determined by the number of cells that can be generated (the 'biomass') and the amount of recombinant biotherapeutic protein that is produced by each cell (the so-called 'cell specific productivity'). The mammalian target of rapamycin (or mTOR) is a master regulator of both cell growth and proliferation (and hence biomass) and of protein synthesis. However, it remains to be established how this master regulator contributes to recombinant protein synthesis, and whether mTOR can be manipulated to enhance growth and product levels. It is therefore surprising that there has been no comprehensive study of the role of mTOR signalling with respect to the role this might play in determining recombinant protein yields from mammalian cells. We will carry out such a study, investigating our overall hypothesis (i) that the growth and productivity of mammalian recombinant cell lines is underpinned by by mTOR, the global regulator of cell proliferation, ribosome biogenesis and protein synthesis, and (ii) that engineering of this global regulator and signalling pathway increases the rate at which cells divide , and enhances recombinant protein production from CHO cells. The overall aim is to generate new mammalian cells that exploit manipulations of mTOR signalling to enhance the production of monoclonal antibodies and other recombinant products. This information is of very substantial relevance to industry since the production of commercially valuable proteins (e.g. antibodies) is hindered when cells become stressed later in culture and by the amount of biomass accumulated in the bioreactor. Without improved expression systems the biotechnology/pharmaceutical industries will lack the capability to produce large enough amounts of these valuable and effective drugs to meet the demand at a price that will allow them to be prescribed for patients who would benefit from them.