Arne Skerra
Technical University, Munich, Germany

Anticalins, Small Antibody-Like Proteins with Engineered Target Specificities

During the past 100 years antibodies have been considered as ideal tools for molecular recognition. The development of methods for the production of their fragments in E. coli [1] has enabled attempts to generate recombinant antibodies by protein engineering. However, with respect to novel applications in biotechnology and medicine it appears that immunoglobulins may not always be optimally suited, due to their composition of two different polypeptide chains and to the comparably large size, even of their functional fragments.

Proteins from the lipocalin family could provide an alternative scaffold [2] for the generation of ligand-receptor proteins by protein engineering. Natural lipocalins are compact proteins, which serve for the transport or storage of certain biomolecular compounds in several organisms. Their tertiary structure comprises a circularly closed eight-stranded anti-parallel b-sheet. This b-barrel supports four loops at one of its ends, which form the entrance to the binding pocket and are structurally variable. In comparison with recombinant antibody fragments lipocalins provide several benefits because they are composed of a single polypeptide chain, have a much smaller size, and their set of four loops can be easily manipulated at the genetic level.

In a combinatorial protein design approach we set out to extensively reshape the ligand pocket of the bilin-binding protein from Pieris brassicae, thus creating artificial ligand-binding proteins termed "anticalins" [3]. A library was prepared by subjecting 16 amino acid positions in the four loops of this prototypic lipocalin to simultaneous random mutagenesis, followed by panning with different immobilized compounds via phage display. In the case of fluorescein as a model hapten, mutants with KD values in the low nanomolar range were selected. Lipocalin variants with specificities for other organic molecules, including the widely applied digoxigenin group [4], peptides, and even protein targets have meanwhile been generated.
Anticalins can be produced at high levels in E. coli or yeast and they exhibit surprising stability, with typical denaturation temperatures above 60°C. They are amenable to the production of functional fusion proteins with enzymes like alkaline phosphatase, which may serve as a convenient reporter enzyme for diagnostic applications. Furthermore, the linking of two anticalins that recognize different ligands results in bispecific binding proteins, termed "duocalins" [5].

Hence, specifically engineered anticalins should not only provide novel reagents for bioanalytics. In principle, they offer advantageous properties for substituting conventional antibodies in other areas [6], for example in cancer therapy. Anticalins may be particularly suitable as small and robust molecular recognition units in drug targeting applications. In order to minimize an immunological response upon therapeutical administration, anticalins derived from a human lipocalin framework are of particular interest in this respect.

References:
[1] Skerra, A. (1993) Bacterial expression of immunoglobulin fragments. Curr. Opin. Immunol. 5, 256-262.

[2] Skerra, A. (2000) Lipocalins as a scaffold. Biochim. Biophys. Acta 1482, 337-350.

[3] Beste, G., Schmidt, F. S., Stibora, T. & Skerra, A. (1999) Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold. Proc. Natl. Acad. Sci. USA 96, 1898-1903.

[4] Schlehuber, S., Beste, G. & Skerra, A. (2000) A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin. J. Mol. Biol. 297, 1105-1120.

[5] Schlehuber, S. & Skerra, A. (2001) Duocalins, engineered ligand-binding proteins with dual specificity derived from the lipocalin fold. Biol. Chem. 382, 1335-1342.

[6] Skerra, A. (2001) "Anticalins": a new class of engineered-ligand-binding proteins with antibody-like properties. Rev. Mol. Biotechnol. 74, 257-275.