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Design of multivalent complexes using the barnase·barstar module

Nature Biotechnology volume 21, pages 1486–1492 (2003)Cite this article

Abstract

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.

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Figure 1: The concept of creating multimeric miniantibodies using heterodimeric barnase·barstar modules and scFv fragments.
Figure 2: The preparation of homogeneous mono- and multivalent proteins.
Figure 3: Assembly of multimeric proteins and their antigen binding.
Figure 4: Comparison of dissociation kinetics of monomeric, dimeric and trimeric 4D5 scFv–constructs measured by surface plasmon resonance (BIAcore).

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Acknowledgements

The authors thank Jörg Willuda for discussions during the initial phase of this project, Annemarie Honegger for molecular modeling, Stephen F. Marino for help and comments, and Frank Bootz and Lydie Chané-Favre for help in the immunogenicity experiments. The work was supported by grants from, among others, the Swiss National Science Foundation (no. 7UPJ062274), the Russian Foundation of Basic Research (no. 01-04-49450) and the Russian Science Support Foundation (no. 2077.2003.4) and PCB RAS.

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Authors and Affiliations

  1. Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry and Institute of Gene Biology, Russian Academy of Sciences, Miklukho-Maklaya str.16/10, Moscow, 117997, Russia

    Sergey M Deyev & Ekaterina N Lebedenko

  2. Center of Radiopharmaceutical Science, Villigen, CH-5232, PSI, Switzerland

    Robert Waibel & August P Schubiger

  3. Department of Biochemistry, University of Zürich, Winterthurer str. 190, Zürich, CH-8057, Switzerland

    Andreas Plückthun

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  1. Sergey M Deyev
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Correspondence to Sergey M Deyev or Andreas Plückthun.

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Deyev, S., Waibel, R., Lebedenko, E. et al. Design of multivalent complexes using the barnase·barstar module. Nat Biotechnol 21, 1486–1492 (2003). https://doi.org/10.1038/nbt916

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