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

抗体是医疗保健中重要的重组分子。它们既是诊断工具，也是治疗方法。它们可以在哺乳动物细胞系中重组表达，因此可以经过特定的蛋白质修饰，从而提高它们的诊断价值或治疗能力。为了表达重组抗体，通常将相应的基因导入细胞系，使其能够高水平表达重组蛋白，并可培养到高密度。需要对转基因细胞系进行分离，并对其表达水平进行彻底分析。这需要广泛的筛查计划，这既耗时又耗费大量费用。此外，重组基因的表达可以通过表观遗传机制来沉默，为了生产重组蛋白，需要在单个细胞克隆中评估转基因表达的稳定性。克服这些限制的方法之一是将编码重组蛋白的基因插入到一个高活性的基因位点。精确整合到一个确定的基因组位置，使转基因具有目标位置的启动子和增强子区域。我们研究了在体外和转基因动物中插入的转基因的调控，发现插入的转基因确实能够采用整合位点的调控模式。因此，我们启动了一个项目(目前由BBSRC资助)，在该项目中，我们结合了同源重组、定点重组和体细胞融合，将转基因整合到典型抗体表达细胞系(如CHO、HEK293和NS0)中高度活跃的基因组位点。现在，我们建议将这种方法扩展到一个整合的噬菌体展示-哺乳动物表达系统，在该系统中，从噬菌体文库中选择的抗体的可变区基因可以通过一步重组过程从噬菌体展示载体转移到哺乳动物细胞系中的预定整合位点。该方法将利用我们实验室分析的异种重组酶靶点。噬菌体展示载体将被设计成使得一对靶点位于重链可变区(VH)的侧翼，另一对LOX位点将位于轻链可变区(VL)的侧翼。相同的靶点对将被放置在典型表达细胞系的高表达基因位点(我们已经鉴定)中。将噬菌体展示载体DNA与位点特异性重组酶的表达载体一起，将VH和VL区置于高表达的区域，这也将提供剩余的重链和轻链蛋白。该项目将包括发展噬菌体展示载体结构和噬菌体展示文库，设计抗体表达结构和表达细胞系。该系统将允许重组抗体在稳定和具有良好特性的哺乳动物表达系统中在数周内在噬菌体展示系统中进行高水平表达。这一战略将大大缩短重组抗体产品的上市时间。经过仔细的验证，这些噬菌体展示系统和表达细胞系将提供有市场价值的实体。该过程及其衍生的细胞系将代表知识产权，在与学术伙伴达成必要协议的情况下，产业伙伴将利用这些知识产权。

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.