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

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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.