Characterization of post-transcriptional constraints that determine rP yield during bioprocessing in mammalian cells

We all have an in-built defence mechanism to respond to infection when our body recognises a foreign 'invader'. A type of cell known as a B cell responds to infection by changing into an antibody-producing cell. Antibodies are proteins that work by attacking the foreign invader and destroying it, clearing infection by removing the foreign agent. Because antibodies are our body's natural defence against disease, many new antibody type drugs are being developed to help treat a number of human diseases such as cancer. These antibodies are usually produced by cells kept in a culturing solution under defined conditions. The problem is that these antibodies must be in a special shape; otherwise they do not work. The cells used to produce such antibodies have a very complex set of machinery to make the antibodies and put their components together into the right shape. This works very well when the cell is not expected to make much of the antibody in question. However, the cells we use to make antibodies are much less efficient at producing these drugs when we try and produce more of the product. As a result, we are not able to produce enough of these drugs and the cost and demand for them is therefore high e.g. the breast cancer drug Herceptin. The root of the problem is that when cells are asked to produce much more protein to meet our needs the machinery can no longer cope / the cells sometimes die or else don't produce antibodies of the right shape, of no clinical use whatsoever. It is largely agreed that this problem will become even serious as further antibody-based drugs are developed. The research proposed here will investigate how the cell machinery for making proteins works and examine whether, and in what ways, it can be manipulated to produce more antibody. We want to determine the different parts of this machinery that are limiting in terms of making the antibody, and then investigate how these parts work together to ultimately produce the antibody of interest. At present it is unknown if this is possible, and the process is poorly understood in the mammalian cells presently used to produce antibodies. We will employ a combination of new state-of-the-art technologies and approaches to take apart the antibody assembly line in mammalian cells in a step-wise manner, and then using the information gathered will determine the relationship between each step of the assembly process. Ultimately this should enable the manipulation of cells to change the balance of each step in the assembly line to produce more of the target antibody drug at reduced cost and higher quality. As stated above, this is extremely important as it is expected that with an increasing number of protein 'drugs' being developed we will lack the capability of producing large enough amounts to meet the required demand for these new drugs for the majority, as opposed to for those who can afford what must currently remain prohibitively expensive, but very effective, medicines.

In recent years the capacity of expression systems (e.g. E. coli, animal cells) to produce recombinant proteins (rP) has advanced through improved growth medium, vector systems and bioprocessing. On-the-other-hand, our understandings of those cellular processes that constrain or limit rP production (rPP) during bioprocessing are poorly understood and open to conjecture. The proposed programme of research will utilise a combination of related approaches to investigate the hypothesis that it is the balance between mRNA levels and their regulatory features, mRNA turnover, the translational machinery, key chaperones, protein folding/degradation, and the cell's ability to coordinate and manipulate each of these processes to the demands of heterologous protein production that ultimately determines recombinant protein expression levels. To achieve this 5 related approaches will be employed. The 1st approach will characterise the diversity of translational machinery within cell populations and the effect of 5'-UTRs on mRNA translation. The 2nd approach will investigate those translational/post-translational mechanisms that limit the quantity and quality of rPP, determining the flux throughout the system by measurement of cell division rates, mRNA and protein levels and half-lives in model expression systems. The 3rd is targeted at the functional and physical link between translation and the chaperone CCT. The 4th approach will investigate the coordination and functional relationship between key members of the chaperone network, protein folding/turnover, and the relationship of these events to rPP. The 5th approach is to investigate disulphide bond formation pathways during protein folding. The proposal provides a rational approach towards understand the limitations and processes/molecular response(s) governing rPP in eukaryotic systems (outcome 1) and for identification of rational targets/strategies for enhancing rPP and for novel gene expression technologies (outcome 2).