title: Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.
abstract: BACKGROUND
  Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen.
  
  
  METHODS
  Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system.
  
  
  RESULTS
  An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 x 10(8) M(-1)) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining.
  
  
  CONCLUSIONS
  ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.
authors.affiliation: ENEA BIOTEC-GEN, CR Casaccia Via Anguillarese 301, 00060 Rome, Italy. galeffi@casaccia.enea.it
authors.forename: Patrizia
authors.initials: P
authors.lastname: Galeffi
authors.forename: Alessio
authors.initials: A
authors.lastname: Lombardi
authors.forename: Immacolata
authors.initials: I
authors.lastname: Pietraforte
authors.forename: Flavia
authors.initials: F
authors.lastname: Novelli
authors.forename: Monica
authors.initials: M
authors.lastname: Di Donato
authors.forename: Maria
authors.initials: M
authors.lastname: Sperandei
authors.forename: Andrea
authors.initials: A
authors.lastname: Tornambé
authors.forename: Rocco
authors.initials: R
authors.lastname: Fraioli
authors.forename: Aline
authors.initials: A
authors.lastname: Martayan
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Natali
authors.forename: Maria
authors.initials: M
authors.lastname: Benevolo
authors.forename: Marcella
authors.initials: M
authors.lastname: Mottolese
authors.forename: Francisco
authors.initials: F
authors.lastname: Ylera
authors.forename: Cristina
authors.initials: C
authors.lastname: Cantale
authors.forename: Patrizio
authors.initials: P
authors.lastname: Giacomini