Towards the manipulation of the UPR in mammalian cells to orchestrate cellular re-organization for enhanced monoclonal antibody production

We have developed a defence mechanism to respond to infection when our body recognises a foreign 'invader'. We have a type of cell known as a B cell in our body, which in response to infection changes into an antibody-producing cell. This is necessary to allow us to fight off infections that would otherwise be very harmful. The antibodies work by attacking the foreign invader and destroying it, thereby clearing the infection and removing the foreign agent. In order for the B cells to change into antibody producing cells (a process known as differentiation), they must adjust the organization of themselves so that they have the required pieces of cellular machinery to make large amounts of antibody. As antibodies are the bodies natural defence against disease, many new antibody type drugs are being developed to help treat human diseases such as cancer and AIDS. However, in order to produce these next generation antibody-based therapeutic 'drugs' we must use mammalian cells to make them. The types of cells we use to make these drugs are not as efficient at producing antibodies as the modified B cell and as a result we are not able to produce enough of these drugs and the cost and demand for them is therefore high. It is thought that this will become even more of a problem as more antibody based drugs are developed. The research proposed here will examine whether we can find mammalian cells with more of the required machinery to produce high-levels of antibodies, or alternatively, if we can manipulate these mammalian cells to produce more of this machinery so that higher yields or amounts of these drugs can be produced more quickly at less cost. At present it is unknown if this is possible, and the process is poorly understood in the mammalian cells used to produce these antibodies. Advanced technology known as proteomics and inducible expression technology will be used to study the differences in the levels of the proteins known to be important for antibody production in differentiated B cells and compare the levels of these proteins in the mammalian cells used for commercial antibody production. We will look for proteins that become either more or less abundant (by altered gene expression, protein synthesis and/or protein degradation) and for subtle molecular modifications to pre-existing proteins known to be able to modify their function (e.g., switch them on or off). Information from current genomics projects will be mined and used in combination with our protein data to identify ways of improving the amount of therapeutic protein 'drug' we can manufacture using these mammalian cells. 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.

In recent years our understanding of the processes by which B cells differentiate into plasma cells, and the changes in the cellular requirements for antibody production and secretion associated with this process, has advanced through the use of 'systems biology' approaches and investigations. Such studies have shown that the differentiation of these cells into 'antibody factories' is dependent upon the regulation of components of the unfolded protein response (UPR). In direct contrast to this, the relationship between the UPR and the rate of monoclonal antibody production or secretion from in vitro cultured mammalian cells are poorly understood and open to wide conjecture, although undoubtedly a better understanding of the detailed mechanistic effects of the UPR on cellular responses in relation to cell specific productivity will provide information allowing the further improvement and optimisation of cells in culture. As such, the exact effect of the UPR and how its control could be managed to improve recombinant protein synthesis in mammalian expression systems is unknown. Therefore, before undertaking the task of global UPR manipulation there is a need to further our fundamental biological knowledge of the UPR and its relationship to recombinant protein production from in vitro cultured mammalian cells. The proposed programme of research will utilise a combination of approaches, including inducible expression technology, cell engineering and advanced proteomic analysis to characterise the URP intracellular architecture associated with high-level recombinant monoclonal antibody production in mammalian cells. Further, we propose to investigate the changes in functional gene expression and protein modification(s) of the UPR that are implicated in the molecular response to the demands of recombinant protein synthesis in cultured mammalian cells by using inducible expression systems to 'impose' and 'alleviate' a UPR in industrially relevant in vitro cultured mammalian cells. The proposal therefore provides a rational approach to understanding the UPR, its relationship to expansion of ER and the rest of the protein secretory apparatus, and how control of UPR (or specific components) can be managed to improve recombinant protein synthesis in mammalian expression systems. To achieve this we will undertake five related approaches. The first approach is targeted at the diversity of UPR protein components in parental cell hosts and cell lines with varying MAb expression, and is pertinent to understanding the relationship between UPR cellular architectural protein markers and the levels of secreted monoclonal antibody. The second is to investigate the relationship between IgG light- and heavy-chain abundance and MAb production. The third is targeted at identifying UPR- and recovery- induced (or down-regulated) proteins and relating this to secreted MAb production, thereby confirming that the cellular requirements for higher qMAb from in vitro cultured mammalian cells are dependent upon the regulation of components of the UPR. The fourth approach will investigate the effect of UPR on translational control, and global and MAb protein expression. The fifth will investigate the effect of XBP1 expression on the global expression of UPR targets and the downstream effects on reporter gene and MAb expression. The outcomes of this research will be (i) an understanding of the molecular response(s) governing UPR adaptation in the industrial environment and the implications for qMAb in in vitro cultured mammalian cells, (ii) the determination of the key UPR architectural components required for enhanced qMAb and the application of this information to create a screen for such traits in parental sub-clones, and (iii) the identification of rational targets for enhanced gene expression technologies.