An integrated cell and protein engineering approach to generate enhanced CHO cell platforms for manufacture of difficult to express biopharmaceuticals

Recombinant biotherapeutics are medicines for diagnosis and treatment of disease. Those of interest in this project are proteins that are built (by cells acting as programmed factories) from knowledge of the body's own macromolecules and are redesigned for the treatment of specific diseases. For example, versions of insulin (for treatment of diabetes) have been designed that have given diabetics much greater control of their health. Other natural molecules (antibodies) are being synthesized to treat cancer and autoimmune diseases. The ability to make biotherapeutics has been driven by industry with the potential for life-changing treatments balanced against the commercial costs of production. A new generation of biotherapeutics (with the potential to treat an increased range of diseases with much greater effectiveness than the existing biotherapeutics) are in development. The new generation is being designed by taking bits of natural molecules and building novel molecules (in modular structures) for production in cell factories. This is what is called synthetic biology, the engineering of new genetic materials for valuable functions. Whilst this has incredible promise and potential, the ability to produce novel biotherapeutics is far from optimal (due to the manner in which the cell factories handle such unnatural products). To harvest the full potential of these novel medicines, there is a need to more fully understand the processes that take place in the cell factory and, with knowledge of those processes, to enhance their production. This project aims to address the fundamentals that will determine the effectiveness of production of novel biotherapeutics and provide new systems that allow the production of these potentially powerful new medicines at a yield and quality not currently possible. In this study, we will investigate these problems using biotherapeutic products that exemplify the pipeline of real potential products under development at UCB (a company that is at the forefront of technologies to manufacture novel biotherapeutics).

Overall, the project aims to investigate and determine how the cell factory responds to the challenge of production of a series of novel biotherapeutics. We also aim to redesign the molecules themselves, by adding into the molecules the ability to have sugars modify specific parts of the molecule, to drive quality control mechanisms in the cell factory that will allow the better assembly and production of the target molecules. We will also use the latest approaches to modifying the cell factory itself, so termed genome editing approaches, to allow us to unravel the 'roadblocks' in the cell factory that prevent production of the molecules of interest and that also allow us to manipulate the cell factory in an attempt to overcome any observed roadblocks. Thus, we will generate an understanding of why the cell factory does not produce difficult to express molecules, how the additional of sugars onto the molecule may help the cell factory recognise 'badly made' material and correct this, and new cell factory systems for industrial application for the manufacture of these new medicines.

In delivering the project we will bring together two world-leading laboratories studying cell factories and their manipulation and the needs of UCB towards production of novel biotherapeutics. The understanding that will arise from the project has wide-scale potential for (i) fundamental understanding of the manner in which cell factories operate and their selective control of production of biotherapeutics of different structure and (ii) for translation of findings into industrial practice (at UCB and wider) for more rapid, more certain and less costly production of new medicines. The approach developed by this project will have widespread value across the entire commercial sector and will have direct relevance to the potential to treat many clinical conditions.

Recombinant produced protein-based biotherapeutics are a huge commercial success, particularly of monoclonal antibodies that dominate the market. As such, the cost (and certainty) of production is relatively fixed for standard antibody-based biotherapeutics and there is predictability and robustness. However, the market is changing and estimates are that 50-60% of all biotherapeutic products in development are non-standard molecules, referred to as novel format (or colloquially as "difficult-to-express") biotherapeutics. Such molecules are generated by application of protein engineering, taking molecular domains and structures of desirable feature (for molecular targeting, functional activity, therapeutic longevity, ease of production) to build a product of non-natural (but potential great efficacy) for production by genetic manipulation of host cell types. The potential for this has not been realized due to low and unpredictable expression of many novel format molecules. This is the focus of the current application. In this project, we will (a) develop a systems level understanding of features that recognise protein quality "acceptability" in particular CHO cell background(s), (b) identify molecular features that enable early selection of "good" clones following transfections, design pipelines for re-engineering CHO cells to generate improved expression of defined novel protein domain builds, (c) develop a greater understanding of the multi-facetted interactive cell background that regulates cell fate, survival and secretion in the industrial context, (d) develop protein engineering strategies, specifically around glycosylation of proteins (and domains) to modify the rate or capacity for secretion of specific difficult to express proteins, and (e) deliver a suite of new CHO cell hosts and protein engineered molecule formats validated for enhanced industrial production and quality of DTE biotherapeutics.

Increased fundamental understanding of cell factories for predictable production of the next generation of novel biotherapeutics will lead to more effective treatment of patients with diseases that may currently be untreatable or without complete cure. The understanding gained (along with the potential to apply synthetic biology approaches to re-engineer the Chinese hamster ovary cell factory or to rationalize the structure of novel biotherapeutics) has immediate implication and long-term potential application. The immediate beneficiaries include the academic and industrial community that is actively addressing approaches for more robust production of novel biotherapeutics. In the longer term, via translation of the research findings into development and manufacturing processes, there will be better therapeutics for patients. Commercially, greater reassurance and, hence, speed to market will decrease the cost of manufacture of new medicines, with implications towards the cost of therapy for health agencies and, ultimately, for the well-being of patient cohorts. The UK is recognized as a leading player (both academically and industrially) across the international sector for research into, and manufacture of, biotherapeutics, a market with sales of $140Bn in 2013. This research, as a collaboration with UCB (one of the world-leading producers of biotherapeutics), will help the UK to maintain its status as an innovator for biotherapeutics. A major outcome of the project will be to highlight the strong industrial-academic collaborative ethos in the UK and the manner in which BBSRC industrial-academic funding initiatives ensure long-term retention of strong UK-based industrial research focus for multi-national companies. At a training level, the PDRAs funded under this LINK project will be embedded into the laboratory environment of UCB for the early stages of the research. They will gain experience of the industrial perspective of direct commercial relevance of the discovery, development and manufacturing focus and will be exposed to the broader UCB environment, participating in project team meetings and attending workshops on technology and enterprise development, drug discovery and commercialization.

Project partners have discussed and accepted a strategy for knowledge exchange and data dissemination. All partners are members of other existing successful collaborations (with a mixture of industrial and academic partners) and have used a range of knowledge exchange pathways to disseminate data and results of potentially commercial-sensitive information in modes that satisfy the needs of all participants (and research funders). A 3-way contract between Kent, Manchester and UCB will develop agreed ownership of intellectual property (IP) and responsibility for its application, ensuring that the support of BBSRC can be acknowledged and that knowledge generated in the project can be disseminated in an appropriate manner. With the contract in place, the project team (and, wherever possible, specifically the PDRA) will disseminate outputs via peer-reviewed publications and at research conferences. This will include UK-based sector networks e.g. BioProNET (http://www.biopronetuk.org) and other Networks in Industrial Biotechnology and Bioenergy (NIBB), bioProcessUK (http://www.bioprocessuk.org) and ESACT UK (http://www.esactuk.org.uk). Internationally, presentations will be made at the two premier sector conferences (ESACT, http://www.esact.org and ECI, Cell Culture Engineering, http://www.engconf.org/conferences/biotechnology/cell-culture-engineering-xv/). The wider Industrial Biotechnology (IB) community will be engaged through the IB Leadership forum (https://connect.innovateuk.org/web/industrial-biotechnology) and we will develop public engagement activities through outreach activities (specifically in Kent and Manchester via the Public Open Days and via the UCB website that has a patient-inspired focus).