Review
Natural and designer binding sites made by phage display technology

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Abstract

In the past decade, the drive to develop completely human antibodies for human therapy has led to the development of phage display technology. This technology is able to deliver the ultimate in antibody engineering, that is, high-affinity fully human antibodies to any antigen of choice. Here, this application of phage display technology is reviewed, and the many other antibody-engineering avenues this technology offers are highlighted.

Section snippets

Making and selecting antibody libraries

The concept of molecular display technology is the provision of a physical linkage between genotype (the antibody variable region genes) and phenotype (antigen-binding) to allow simultaneous selection of the genes that encode a protein with the desired binding function. This concept was successfully applied to small peptides by Smith5, but the display of functional antibody repertoires on phages required several additional discoveries. First, a procedure for accessing large collections of

Selection for binding using purified antigen

The selection step is a key feature of any molecular display technology. A major advantage of this in vitro procedure is that we can choose and manipulate the procedure and therefore the outcome of the selection (reviewed in Ref. 30). Selections with purified antigen are essentially made in two ways. The antigen can be attached to a solid support (to plastic, by adsorption, or to beads or a column matrix), the phage library run over the support, and antigen-bound phages retrieved after rinsing

Exploring the natural binding site

Binding sites evolved by the immune system might be rescued from immune hosts and cloned using phage-display technology, as has been done for mice, rats, rabbits, camels and humans. In humans, phage antibody libraries made from donors who naturally mount an immune response (with viral infections, bearing tumours or with autoimmune disease) have been used to generate both human antibodies with high affinity and specificity for particular antigens, and to investigate the humoral response in

Improving the binding site

The antibodies selected from many of the immune and the large ‘single pot’ repertoires are often directly useful for the researcher, for example for ELISA, western blot or immunofluorescence. In some instances, their affinity is not sufficient, such as for many diagnostic applications or some applications in immunotherapy. Affinity maturation might be bypassed by the construction of multivalent molecules, as reviewed in 49, 50; however, there will be occasions in which in vitro affinity

Exploring the designer binding site

Binding-site repertoires with naturally rearranged V-genes are potentially heavily biased towards certain sequences and clones, owing to evolutionary pressures and the immune history of the host. This will result in biases and redundancy in natural binding-site libraries (Table 1). The use of laboratory-assembled variable-region genes would provide complete control over the genetic makeup, allow engineering of the individual building blocks, and a choice of where and how the diversity could be

Smaller Ig-fold-based binding sites

Nature has evolved to use two protein domains with an immunoglobulin fold in nearly all of its natural antigen combining sites. Nevertheless, engineering of binding sites based on a single variable domain with immunoglobulin fold8 might yield ligands with specific applications that antibodies with two variable domains cannot offer. Several single-domain antibody formats have been explored with phage display, including the naturally occurring heavy-chain only antibodies from camels66, as well as

Phage display-based human therapeutic antibodies

Where antibodies have, however, a very strong competitive advantage, is in their use as reliable, relatively easy to produce, well-tolerated and well-behaved immunotherapeutics73. In this regard, immunogenicity has proved to be a major drawback in clinical applications of antibodies, thus for most therapeutic antibodies one would prefer a fully human antibody sequence. Less than ten years after the description of the concepts, the first phage display technology-derived antibodies, made by

Concluding remarks

Our efforts to make antibodies without using animals and outside the immune system have now provided us with very powerful tools for in vitro selection and evolution of binding-site libraries. Significant advances in the past decade with phage and other display methodologies, binding-site library design, refined selection procedures and instrumentation for automation, are making display technologies increasingly popular for creating binding sites for use in all areas of research, and in medical

Acknowledgements

We gratefully acknowledge S. Hufton and P. Henderikx for critically reading the manuscript.

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