Abstract
We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained aganist five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Huang, W., Dolmer, K. & Gettins, P.G. NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein. J. Biol. Chem. 274, 14130–14136 (1999).
North, C.L. & Blacklow, S.C. Structural independence of ligand-binding modules five and six of the LDL receptor. Biochemistry 38, 3926–3935 (1999).
Gliemann, J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands. Biol. Chem. 379, 951–964 (1998).
Krieger, M. & Herz, J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu. Rev. Biochem. 63, 601–637 (1994).
Rettenberger, P.M. et al. Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein. J. Biol. Chem. 274, 8973–8980 (1999).
Mammen, M., Choi, S.K. & Whitesides, G.M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2755–2794 (1998).
Koduri, V. & Blacklow, S.C. Folding determinants of LDL receptor type A modules. Biochemistry 40, 12801–12807 (2001).
Physicians Desk Reference, edn. 59 (Medical Economics, Montvale, N.J., 2005).
Suffredini, A.F., Fantuzzi, G., Badolato, R., Oppenheim, J.J. & O'Grady, N.P. New insights into the biology of the acute phase response. J. Clin. Immunol. 19, 203–214 (1999).
Chang, C.C. et al. Evolution of a cytokine using DNA family shuffling. Nat. Biotechnol. 17, 793–797 (1999).
Crameri, A., Raillard, S.A., Bermudez, E. & Stemmer, W.P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).
Patten, P.A. & Schellekens, H. The immunogenicity of biopharmaceuticals. Lessons learned and consequences for protein drug development. Dev. Biol. (Basel) 112, 81–97 (2003).
Binz, H.K. & Pluckthun, A. Engineered proteins as specific binding reagents. Curr. Opin. Biotechnol. 16, 459–469 (2005).
Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. & Heyneker, H.L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).
Stemmer, W.P.C. in The Encyclopedia of Molecular Biology, 447–457, (VCH Publishers, New York, 1996).
Szybalski, W., Kim, S.C., Hasan, N. & Podhajska, A.J. Class-IIS restriction enzymes–a review. Gene 100, 13–26 (1991).
Coulie, P.G., Stevens, M. & Van Snick, J. High- and low-affinity receptors for murine interleukin 6. Distinct distribution on B and T cells. Eur. J. Immunol. 19, 2107–2114 (1989).
Hammacher, A. et al. Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. Protein Sci. 3, 2280–2293 (1994).
Acknowledgements
The authors would like to thank Evan Green, Cynthia Bailey, Kevin Allen, Anielka Montalvan and Khue Dang for their expert laboratory assistance, Jeff Smith and Peter van Vlasselaer for their assistance in overall project management, all the contributors at Boehringer-Ingleheim Austria for their work in fermentation and production scale-up, and Harry Ruan of Comparative Biosciences for performing the in vivo portion of the experiment shown in Figure 3.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Fig. 1
Alignment of human A-domain sequences. (PDF 130 kb)
Supplementary Fig. 2
Pharmacokinetics of an avimer in cynomolgus. (PDF 55 kb)
Supplementary Fig. 3
Surface plasmon resonance analysis of C326 binding to IL-6 immobilized to a CM-5 chip. (PDF 118 kb)
Supplementary Fig. 4
Avimers are not immunogenic in mice. (PDF 81 kb)
Rights and permissions
About this article
Cite this article
Silverman, J., Lu, Q., Bakker, A. et al. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 23, 1556–1561 (2005). https://doi.org/10.1038/nbt1166
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nbt1166
This article is cited by
-
Biomolecular sensors for advanced physiological monitoring
Nature Reviews Bioengineering (2023)
-
In vitro-engineered non-antibody protein therapeutics
Protein & Cell (2018)
-
Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity
Scientific Reports (2017)