title: In vitro evaluation of avidin antibody pretargeting using 211At-labeled and biotinylated poly-L-lysine as effector molecule.
abstract: BACKGROUND
  Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled poly-L-lysine was constructed and analyzed in vitro.
  
  
  METHODS
  Avidin activated by 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and thiolated trastuzumab were incubated overnight at 4 degrees C. The monomeric fraction was extracted using size exclusion fast protein liquid chromatography (FPLC) and further purified on an iminobiotin affinity column. Poly-L-lysine was biotinylated with succinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin), followed by direct (211)At-labeling with N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), and succinylation with succinic anhydride. The avidin-trastuzumab conjugate was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and FPLC, together with cell-binding and biotin-binding analyses. The labeled poly-L-lysine conjugate was assessed in terms of radiochemical purity and avidin binding. Furthermore, the full pretargeting system was evaluated in a tumor cell binding assay.
  
  
  RESULTS
  The estimated size of the pretargeting molecule was 220 kDa, which corresponds to that of the expected avidin-trastuzumab monomer. Neither cell-binding ability (64%) nor biotin-binding ability (85%-95%) indicated any severe adverse effects from the chemical modifications. The radiochemical purity of the effector molecule was 92%-97%, and the avidin binding capacity was 91%-93%. The complete pretargeting assay resulted in a binding of 75.3 +/- 6.2% of added effector molecules to cells.
  
  
  CONCLUSIONS
  The high binding of effector molecules to cells demonstrates a proof of concept for the synthesized molecules and pretargeting system, which will be further evaluated in vivo in future studies.
authors.affiliation: Department of Radiation Physics, Sahlgrenska Academy, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden. sofia.frost@radfys.gu.se
authors.forename: Sofia H L
authors.initials: SH
authors.lastname: Frost
authors.forename: Holger
authors.initials: H
authors.lastname: Jensen
authors.forename: Sture
authors.initials: S
authors.lastname: Lindegren
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D001246:Astatine / Q000627:therapeutic use*; D001360:Avidin / Q000008:administration & dosage*; D019921:Biotinylation; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D010455:Peptides / Q000737:chemistry / Q000627:therapeutic use*; D016499:Radioimmunotherapy / Q000379:methods*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D010455:Peptides; D001360:Avidin; C027819:polyleucine; D000068878:Trastuzumab; D001246:Astatine