An integrated phage-display mammalian-expression system for the rapid production of recombinant antibodies

Antibodies are important recombinant molecules in healthcare. They serve as diagnostic tools and as therapeutics. They can be expressed recombinantly in mammalian cell lines and can therefore be subjected to specific protein modifications which increase their diagnostic value or therapeutic potency.

In order the express recombinant antibodies the corresponding genes are typically transfected into cell lines which enable high levels of recombinant protein expression and can be grown to high densities. The transfected cell lines need to be isolated and thoroughly analysed for expression levels. This requires extensive screening programmes which are time consuming and cost intensive. In addition recombinant gene expression can be silenced by epigenetic mechanisms and stability of transgene expression needs to be assessed in individual cell clones for recombinant protein production.

One route to overcome these limitations is the insertion of the gene encoding the recombinant protein into a highly active gene locus. A precise integration into a defined genomic site equips the transgene with the promoter and enhancer regions of the targeted site. We have studied the regulation of transgenes inserted in vitro and in transgenic animals and found that inserted transgenes are indeed able to adopt the regulatory patterns of the integration site. We have therefore initiated a project (currently funded by the BBSRC) in which we combine homologous recombination, site-specific recombination and somatic cell fusion to integrate transgenes into genomic loci highly active in typical antibody expression cell lines (like CHO, HEK293 and NS0).

We now propose to extend this approach to develop an integrated phage display-mammalian expression system in which the variable region genes of an antibody selected from a phage library can be transferred in a one-step recombination process from the phage display vector into a predefined integration site in a mammalian cell line. The approach will make use of heterospecific recombinase target sites analysed in our lab. The phage display vector will be designed such that one pair of target sites will flank the heavy chain variable region (VH) and another pair of lox sites will flank the light chain variable region (VL). Identical pairs of target sites will placed into a highly expressed gene loci (which we have already identified) of a typical expression cell line. Transfection of phage display vector DNA together with an expression vector for the site-specific recombinase will place the VH and VL regions into highly expressed loci which will also provide the remainder of the heavy and light chain proteins. The project will include the development of phage display vector constructs and phage display libraries, the design of antibody expression constructs and expression cell lines.

The system will allow the high-level expression of recombinant antibodies in a stable and well characterised mammalian expression system within weeks of being identified in the phage display system. This strategy will significantly cut the time to market for recombinant antibody products. After careful validation these phage display systems and expression cell lines will provide marketable entities. The process and the cell lines derived from it will represent intellectual property that will be utilised by the industrial partner subject to the necessary agreements with the academic partner.