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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

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and _in vitro_ analyses of multivalent antibodies. _J. Immunol._ 170, 4854–4861 (2003). Article  CAS  Google Scholar  Download references 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 * Center of

Radiopharmaceutical Science, Villigen, CH-5232, PSI, Switzerland Robert Waibel & August P Schubiger * Department of Biochemistry, University of Zürich, Winterthurer str. 190, Zürich,

CH-8057, Switzerland Andreas Plückthun Authors * Sergey M Deyev View author publications You can also search for this author inPubMed Google Scholar * Robert Waibel View author publications

You can also search for this author inPubMed Google Scholar * Ekaterina N Lebedenko View author publications You can also search for this author inPubMed Google Scholar * August P Schubiger

View author publications You can also search for this author inPubMed Google Scholar * Andreas Plückthun View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHORS Correspondence to Sergey M Deyev or Andreas Plückthun. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY FIG. 1 (PDF 137 KB) SUPPLEMENTARY METHODS (PDF 162 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received:

25 August 2003 * Accepted: 25 September 2003 * Published: 23 November 2003 * Issue Date: 01 December 2003 * DOI: https://doi.org/10.1038/nbt916 SHARE THIS ARTICLE Anyone you share the

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