title: Novel preparation and characterization of a trastuzumab-streptavidin conjugate for pre-targeted radionuclide therapy.
abstract: BACKGROUND
  This study describes a novel and convenient route for the preparation of a trastuzumab-streptavidin conjugate such as might be used in a pre-targeting system and its in-vitro and in-vivo evaluation.
  
  
  METHODS
  Trastuzumab was irradiated with UV light in the presence of stannous ions to reduce a number of the disulfide bridges to free thiol groups. A range of irradiation times were studied in order to quantify the number of thiols produced and to optimize the reduction process. The conjugate was then prepared by reaction with succinimidyl 4-(N-maleimidomethy cyclohexane)-1-carboxylate (SMCC)-linked streptavidin.
  
  
  RESULTS
  Initial conjugation reactions in phosphate buffer were inefficient, producing low conjugate yields, but conjugation reactions in triethanolamine-based buffer showed greatly increased conjugation yields. A high purity product (approximately 100%) was obtained following purification by gel-filtration HPLC as determined by subsequent size exclusion HPLC analysis. The conjugate was shown to possess an essentially identical immunoreactivity to that of the native, unconjugated antibody and an unaltered biotin binding stoichiometry. Shedding and internalization by Her-2-expressing cells were low and the uptake in vivo by Her-2-expressing xenografts in nude mice was similar to that of labelled antibody.
  
  
  CONCLUSIONS
  Our results demonstrate a new, simple and effective method for the successful synthesis of antibody-streptavidin conjugates which could also be applied to many other heterodimeric protein conjugation reactions.
authors.affiliation: Cancer Research UK, Nuclear Medicine Group, St Bartholomew's Hospital, London, UK.
authors.forename: James E S
authors.initials: JE
authors.lastname: Hainsworth
authors.forename: Peter
authors.initials: P
authors.lastname: Harrison
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Mather
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D001943:Breast Neoplasms / Q000378:metabolism* / Q000532:radiotherapy; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D007553:Isotope Labeling; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009928:Organ Specificity; D016499:Radioimmunotherapy / Q000379:methods; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000627:therapeutic use; D019809:Streptavidin / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D014018:Tissue Distribution; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; D019809:Streptavidin; D000068878:Trastuzumab