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Combinatorial libraries and phage display

Combinatorial library technology in combination with selection principles like phage display technology has since its introduction in the mid 1980-ies (Smith, 1985) revolutionized the development of biomolecules.

Phage display is an elegant approach whereby the products of a gene harbored within the genome of a bacterial virus (a phage) or a so-called phagemid vector (a plasmid that fools the virus system to believe that it is a viral genome) will be found on the surface of the virus particle. In this way, the genes encoding the protein product will be found inside the virus particle while the function encoded for by the gene (such as the binding properties of an antibody) will be displayed on the surface of the virus.

This combination has two attractive features. Firstly it allows us to select for the properties of the protein on the virus surface. Secondly we can analyze the sequence of the gene found inside the particle that on its surface carried the protein with a desirable property, like binding to a toxin, a cancer cell or a bacterium.

This approach has two important advantages. Firstly, the function (for example the binding properties) of the protein encoded for by the gene cannot be itelf be easily predicted from the gene sequence. Secondly, the sequence of a protein with a desirable function cannot easily be predicted from the function itself. Rather the combined set provides critical complementary sets of information, namely function of the protein and sequence of the gene.

Through the realization that these features could be put together in a single concept as evidenced by phage display (Smith, 1985) we gained access to a tool that allows us to develop proteins with specific binding properties at a rate exceeding that of other technologies.

Initial efforts were focused on the development of peptides with specific binding properties (Smith, 1985). Developments in the field of protein engineering, in particular

  • the realization that heterologous complex proteins like antibody fragments can be produced in functional form in E. coli and secreted from the cell using e.g. pelB leader sequences
  • the realization that diverse antibody-encoding gene sequences could be amplified by PCR (polymerase chain reaction) using a restricted set of primers (Larrick et al., 1989; Orlandi et al., 1989)

paved the way for similar efforts to identify specific binding proteins from large combinatorial libraries.

Altogether these developments were critical for the work that eventually resulted in the first description of a selection process of displayed proteins on the surface of filamentous bacteriophage (McCafferty et al., 1990).

Since then phage display and other technologies like ribosome display (Hanes and Plückthun, 1997), bacterial display (Georgiou et al., 1997), yeast display (Boder and Wittrup, 1997) have been fine-tuned to deliver proteins with very defined properties. Numerous combinatorial libraries have been made either directed towards defined antigens or larger, "naïve" libraries suitable for essentially any type of target (as exemplified by de Haard et al., 1999; Griffiths et al., 1994; Knappik et al., 2000; Løset et al., 2005; Marks et al., 1994; Nissim et al., 1994; Pini et al., 1998; Sheets et al., 1998; Söderlind et al., 2000; Vaughan et al., 1996).

Proteins selected from antibody libraries displayed on phage particles are already in clinical use (Humira®) and many more are undergoing clinical trials for use in the treatment of e.g. cancer, infectious disease and autoimmune disease. Thus this technology has proven its capacity to deliver proteins of biomedical importance.



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