Review articleAntibody phage display technology and its applications
Introduction
The generation of new drugs has long involved the search amongst hundreds of thousands of components using well defined in vitro screening tests, the output of which was chosen to mimic as closely as possible the desired in vivo activity of the new drug. Now new library methodologies offer many alternative and at least as powerful routes, by combining the generation of billions of components with a fast screening or selection procedure to identify the most interesting lead candidates. One of the most widely used library methodologies is based on the use of filamentous phage [1], a bacteriophage that lives on Escherichia coli. Phage-display has proven to be a very powerful technique to display libraries containing millions or even billions of different peptides or proteins. One of the most successful applications of phage display has been the isolation of monoclonal antibodies using large phage antibody libraries [2]. Indeed, in the last few years, very efficient techniques have been developed to design and build large libraries of antibody fragments, and ingenious selection procedures have been established to derive antibodies with the desired characteristics. Here, we review the progress made in this rapidly developing field, and discuss a broad range of applications, including the use of large phage antibody libraries to discover novel therapeutic targets and methods for selection of biologically active ligands. Finally, we address the potential of combining phage display with complementary technologies, to increase the scope and range of applications of this technology.
Section snippets
The phage display principle
The power of the phage display system is illustrated in Fig. 1. DNA encoding millions of variants of certain ligands (e.g. peptides, proteins or fragments thereof) is batch-cloned into the phage genome as a fusion to the gene encoding one of the phage coat proteins (pIII, pVI or pVIII). Upon expression, the coat protein fusion will be incorporated into new phage particles that are assembled in the bacterium. Expression of the fusion product and its subsequent incorporation into the mature phage
Antibody libraries
One of the most successful applications of phage display has been the isolation of monoclonal antibodies from large phage antibody libraries (Fig. 3). We will discuss the three types of such phage libraries, immune, naı̈ve and synthetic antibody libraries.
Diversity in selection methods
Phage antibody selections involve the sequential enrichment of specific binding phage from a large excess of non-binding clones. This is achieved by multiple rounds of phage binding to the target, washing to remove non-specific phage and elution to retrieve specific binding phage (a schematic outline is depicted in Fig. 4). Any method that separates clones that bind from those that do not, can be used as a selection method, and as such, many different selection methods have been used. In Fig. 4
Basic screening assays
The outcome of any selection procedure is a mixture of binding ligands with differing properties. It may be necessary to screen large numbers of antibodies to identify those variants with the most optimal characteristics. The best screening assays are fast, robust, amenable to automation (e.g. 96-well format), and use unpurified phage antibodies, or the soluble antibody fragments from the bacterial supernatant. The screening assay should also be linked as closely as possible to the ultimate
Selection for function
With large libraries at hand, we may go beyond the in vitro binding interaction itself, and select for a particular function. For example, provided reporter systems with sufficient sensitivity are used, it may eventually be possible to sort cells which have been triggered by a phage particle displaying an a(nta)gonistic ligand. Such sorting procedures could allow the direct selection of phage particles with agonist or antagonist activity for a given receptor directly from the phage library.
Beyond antibodies
The ideas on functional selections and screening procedures may be expanded to the use of alternative proteins or protein domains for constructing binding molecules (reviewed in [131]). Scaffolds different from antibodies have been reported to form suitable binding ligands for many types of molecules. There are ample examples of `host scaffolds' that contain sufficient permissive regions to accommodate a reasonable numbers of substitutions, which may be used to generate a library of localized
Perspectives
This review highlights the advantages and possible applications of phage display for the development of antibodies. With this technology, antibody engineering for the first time may be used to design antibodies from scratch, with an option to choose its building blocks, its affinity (up to the picomolar range), its format (size and valency) and its effector function (natural (IgG) or novel (enzymes etc.)) [148]. Tailor-made reagents may thus be generated, for in vitro or in vivo diagnosis and
Acknowledgements
This work has been supported by grants from the Profileringsfonds of the University Hospital Maastricht (grant PF37), the European Community, Biotechnology Programme 5.1 (PL950252), and receives financial support from The Netherlands Technology Foundation (STW) in a project coordinated by the Life Sciences Foundation (SLW) (project 805.17.753).
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