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

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Mead EJ (2014) Control and regulation of mRNA translation. in Biochemical Society transactions

 
Description This project investigated the development of mammalian cell lines for high level expression of recombinant proteins. Although there have been many advances in this field, the time and cost to develop such cell lines, and the enhancement of product yields, remain key areas that require further development in an industrial sense to reduce the timelines/cost of generating cell lines and improve the cell specific and volumetric productivities (total amount of protein drug produced). One such approach to tackling these issues is via cell engineering systems that utilise our ever-improving understanding of the biology underpinning cell growth and recombinant protein synthesis, folding, assembly and secretion. With regard to this, the manipulation of the mTOR (the mammalian target of rapamycin) pathway, a master regulator of protein synthesis, ribosome biogenesis and cell proliferation, has recently been reported to influence cell growth and recombinant protein synthesis from mammalian cells. Protein synthesis and ribosome biogenesis are key determinants of cellular biomass and protein productivity. However, the roles that this signalling pathway plays in underpinning these phenotypes, and whether it can be manipulated to enhance them, have yet to be fully elucidated. We have investigated the role of mTOR signalling in determining the cellular phenotype of recombinant mammalian cell lines and whether this signalling system, and the environmental cues to which it responds, may be manipulated to enhance such phenotypes.

We have shown that mTOR signalling differs between cell lines of different productivities and are currently following this up with regard to whether this knowledge can be used to screen for high producing and growing cell lines. We have also shown that manipulation of key components of this signalling system, specifically a protein involved in elongation of polypeptides during protein synthesis called eEF2, can be used to enhance cell grow and productivity. Finally, we have also established that a key parameter of mTOR signalling is the ratio of specific mTOR components to each other (eIF4E and eIF4E-BP) and that these underpin cell growth and recombinant protein drug yields from mammalian cells.
Exploitation Route As stated above, this project is still in progress. However, the key findings to date can be used to help define new approaches to engineer mammalian cells to yield cells that grow faster and make more recombinant biotherapeutic proteins of higher quality for use in the clinic. We anticipate that industry will be able to evaluate these findings in the industrial setting to determine if these findings can be translated to use in the commercial environment.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The key findings to date have been used in discussions with industry to define cell properties that underpin performance commercially and in the consideration of media required for supporting high cell growth and recombinant protein productivity. The findings have contributed to new funding to commercialise a process in collaboration with an industrial partner - this being a process to enhance the yield and quality of recombinant biotherapeutic proteins produced from cultured mammalian cells, specifically Chinese hamster ovary (CHO) cells. The findings have also been used in outreach activities, particularly explanations to secondary school students undertaking the MBP2 project and demonstrating molecular techniques to Grammar school sudents (Simon Langton Grammar School- Canterbury) by Dr Josse.
First Year Of Impact 2014
Sector Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description MC Training Network EU - Horizon 2020 MSCA
Amount € 819,863 (EUR)
Funding ID ITN 642663 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 03/2015 
End 03/2019
 
Description Royal Society Industrial Fellowship
Amount £173,850 (GBP)
Funding ID IF130004 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 11/2013 
End 10/2017
 
Description Collaboration with South Australian Health and Medical Research Institute Limited 
Organisation South Australian Health and Medical Research Institute
PI Contribution Further investigation of mTOR signalling and impact on recombinant biotherapeutic protein production from Chinese hamster ovary (CHO) cells.
Collaborator Contribution Generation of mutants of mTOR signalling targets.
Impact A patent is currently being drafted.
Start Year 2017
 
Description 7 open days at University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Describing the research we undertake in the laboratory, covering all aspects of the research and the impact this has/can have. Particular questions around genetic modification of cells to produce recombinant proteins in all sessions and discussions around both the ethical aspects of this and the potential applications of such technology.
Year(s) Of Engagement Activity 2015,2016
 
Description Lyne Josse at Simon Langton Grammar School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Questions from students on research work.

Return visits as part of interaction between the School and Kent.
Year(s) Of Engagement Activity 2011,2012,2013,2014
URL http://www.thelangton.org.uk/mbp/
 
Description Open Days At University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity is take take parents and secondary students around my research laboratories to explain the research undertaken and to demonstrate some of the research that we do in the laboratory.

Parents and students asked about engineering of cell lines and therapeutic recombinant protein drugs and how these are made, cost implications and on-going research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Presentation on Biosimilar protein based biotherapeutic drugs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact The talk stimulated discussion with the nurses and health care professionals present about what biosimilars are and when/how to assess if these are appropriate to administer as opposed to the innovator drug.

Nurses and healthcare professionals asked for further information on biosimilars.
Year(s) Of Engagement Activity 2014
 
Description School visit/outreach for several days at Simon Langton Boys Grammar - MBP2 project showing students how to clone, express and purify recombinant proteins in the laboratory and discuss science behind this. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Helping with research activity in school - cloning and expression of recombinant proteins
Year(s) Of Engagement Activity 2015,2016
 
Description Turkey Public Biotechnology Talk 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation to public audience in Turkey on behalf of the British Council around biotechnology and its application. Event was filmed and followed by a question and answer session, answering questions sent in before the talk by social media and then from the audience. The event was filmed and shown on national TV in Turkey. Large range of topics discussed around the application of biotechnology to every day life and issues with long discussion/debate.
Year(s) Of Engagement Activity 2016
URL http://www.britishcouncil.org.tr/en/programmes/education/science-innovation-talks/biotechnology