Design of multivalent complexes using the barnase·barstar module
Sergey M Deyev1, Robert Waibel2, Ekaterina N Lebedenko1, August P Schubiger2 & Andreas Plückthun3
1. Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry and Institute of Gene Biology, Russian Academy of Sciences, Miklukho-Maklaya str.16/10, 117997 Moscow, Russia.
2. Center of Radiopharmaceutical Science, CH-5232 Villigen PSI, Switzerland.
3. Department of Biochemistry, University of Zürich, Winterthurer str. 190, CH-8057 Zürich, Switzerland.
Correspondence should be addressed to S M Deyev. e-mail: deyev@ibch.ru and A Plückthun. e-mail: plueckthun@bioc.unizh.ch
The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185HER2-ECD 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.
Published online: 23 November 2003, doi:10.1038/nbt916
December 2003 Volume 21 Number 12 pp 1486 - 1492 |
