Design of multivalent complexes using the barnase*barstar module.
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-p185(HER2-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.
Deyev
Sergey M
SM
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry and Institute of Gene Biology, Russian Academy of Sciences, Miklukho-Maklaya str.16/10, 117997 Moscow, Russia. deyev@ibch.ru
Waibel
Robert
R
Lebedenko
Ekaterina N
EN
Schubiger
August P
AP
Plückthun
Andreas
A
D000818:Animals; D001426:Bacterial Proteins; D019281:Dimerization; D046911:Macromolecular Substances; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000378:metabolism*; D009928:Organ Specificity; D011485:Protein Binding; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000097:blood / Q000493:pharmacokinetics; D012260:Ribonucleases / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*; D014018:Tissue Distribution
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011506:Proteins; D011993:Recombinant Fusion Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease