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

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.

We will define the role of the protein kinase 'the mammalian target of rapamycin' (mTOR) in determining/limiting recombinant protein (rP) synthesis from mammalian cells and then use this information to generate new host cells with enhanced growth and productivity characteristics via novel mTOR cellular engineering. mTOR is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, processes which underpin rP yield from mammalian cell expression systems, yet the role of mTOR signalling in determining the cellular phenotype of recombinant cell lines and whether this signalling system, and the environmental cues to which it responds, may be manipulated to enhance such phenotypes remain open to question. The work programme will test the hypothesis that 'the growth and productivity of mammalian recombinant cell lines is underpinned by the signalling pathways activated by mTOR, the global regulator of cell proliferation, ribosome biogenesis and protein synthesis, and that engineering of this global regulator and signalling pathway decreases cell doubling times/increases proliferation rates and enhances rP production (rPP) from CHO cells'. We will characterise the links between mTORC1 signalling, culture environment, cell growth/proliferation, ribosome biogenesis, energy transduction, mRNA translation factors and the control of their activity, and rPP. The outcomes of this research will be (i) defining the role of mTOR on controlling mRNA translation and IVC and the contribution of this control to underpinning rPP, (ii) an understanding of the relationship between mTOR signalling, mitochondrion function and rPP, (iii) via cell engineering of mTOR and its upstream and downstream regulators the generation of new CHO host cell lines with reduced doubling time, higher IVC, enhanced mRNA translation and qP, and subsequently increased rP yields, and (iv) development of screening strategies to select for cell lines with enhanced mTORC1 signalling.

Who will benefit from this research?
In terms of research findings, the primary beneficiaries will be researchers in the academic and biopharmaceutical sectors who are interested in understanding the role of the environment and mTORC1 signalling with respect to cell growth, proliferation and recombinant protein synthesis in industrially relevant cell lines. As such, this proposal is relevant to all those academics and industrialists who are interested in the process and/or manufacturing of proteins and wish to deliver them at increased yield in a functionally active form at less cost. The impacts of this research will therefore be national and international and will benefit the following: (1) those in the research fields of cell biology, mTOR signalling and protein synthesis (mRNA translation); (2) the academic and industrial bioprocessing and scientific communities; (3) the biopharmaceutical sector; ultimately the National Health Service (and thus the wider public, its patients) and the UK economy through the development of new methods to produce larger amounts of increasingly important 'bio-drugs' (i.e. recombinant proteins) more efficiently and thus at lower cost.

How will they benefit?
The major impact of this work will be to provide both industry and academia with a much better understanding of the roles of mTORC1 signalling in the production of recombinant proteins in mammalian cells, specifically in industrially-relevant Chinese hamster ovary cells (CHO), and the subsequent application of this information to generate new tools and methodologies (engineered cell lines and alternative feeding strategies based upon amino acid use and mTORC1 signalling). This will allow the faster development of cell lines expressing recombinant proteins at enhanced productivities, lowering the cost of producing such biomedicines. It will also provide both academics and industrialists with a better understanding of the mTOR signalling pathway at a basic biological level in an industrial sense. The ability to produce these high cost drugs at lower cost will ultimately allow access to these drugs to a wider sector of the population both nationally and internationally, thus contributing to health and quality of life. In order to ensure that these delivered, our results will be published in peer-reviewed high-quality journals and presented at relevant conferences. We will publicise our findings through our websites, press releases, BBSRC Business and via the local media and our own public engagement activities, including national and local science fairs and working with local schools. As the PIs are well-placed to inform the activities of industry and to exploit their own discoveries commercially we will build on these industrial links to translate our findings into applications in the recombinant protein production field and inform industry of our results. The PIs, together with Kent Innovation and Enterprise (KIE) and Southampton Research and Innovation Services will take the lead in ensuring this is completed in a timely fashion such that the Universities' IP is protected. KIE would also have the task of determining the market for any IP and initiating dialogue with additional potential collaborators interested in accessing IP or knowhow. Regular teleconferences and meetings between PIs and PDRAs will ensure close coordination between the activities at Kent and Southampton, such that findings in one lab are rapidly conveyed to the other to inform and develop the project in a timely and efficient way. This is detailed further in our pathways to impact document.

This research programme provides ample opportunities for staff training through (i) the range of approaches and techniques to be used; (ii) the close interactions with members of the applicant's laboratories working on projects in similar areas, (iii) interactions with the bioprocessing and pharma industries, and (iv) the opportunity to undertake public engagement work.