title: Evaluation of 177Lu-DOTA-labeled aglycosylated monoclonal anti-L1-CAM antibody chCE7: influence of the number of chelators on the in vitro and in vivo properties.
abstract: BACKGROUND
  In this study, we optimized the 1,4,7,10-tetraazacyclododecane-N-N'-N"-N"'-tetraacetic acid (DOTA) chelator-to-antibody (c/a) ratio for the aglycosylated variant of the anti-L1-CAM antibody chCE7 (chCE7agl), providing high specific activity and low liver uptake in 177Lu-labeled form.
  
  
  METHODS
  chCE7agl was substituted with increasing molar excess of DOTA-NCS. The number of chelators coupled to the antibody and the binding affinities to target tumor cells (IC50 values) of the resulting immunoconjugates were determined. The different immunoconjugates were labeled with 177Lu; specific activity was measured, and metabolic stability was analyzed in human plasma. The effect of different c/a ratios on blood clearance and liver uptake was tested in nude mice. Changes of the protein backbone structure were analyzed by circular dichroism spectroscopy.
  
  
  RESULTS
  chCE7agl was substituted with 7, 12 or 15 DOTA ligands. The IC50 concentrations displacing radioiodinated chCE7 antibody increased with the number of chelators (1.5-fold with 7 ligands, 2.5-fold with 12 ligands and a 5-fold increase with 15 ligands). The highest specific activity for 177Lu-DOTA-chCE7agl was obtained with a c/a ratio of 12 (106 MBq/mg). Radioimmunoconjugates were stable in human plasma for at least 24 h. Blood clearance and liver uptake were measured after 24 h (c/a ratios of 12 and 15) or 48 h (c/a ratio of 7). The liver-to-blood ratios were 0.35+/-0.14 (7 ligands), 0.77+/-0.19 (12 ligands) and 17.85+/-3.44 (15 ligands).
  
  
  CONCLUSIONS
  DOTA-chCE7agl conjugates with a c/a ratio of 12 combined high specific activity with good in vitro and in vivo properties. The rapid elimination rate from the blood and the high uptake in the liver of chCE7agl substituted with 15 DOTA ligands were found not to be due to conformational changes of the antibody backbone structure.
authors.affiliation: Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, CH-5232 Villigen, Switzerland.
authors.forename: Karin
authors.initials: K
authors.lastname: Knogler
authors.forename: Jürgen
authors.initials: J
authors.lastname: Grünberg
authors.forename: Ilse
authors.initials: I
authors.lastname: Novak-Hofer
authors.forename: Kurt
authors.initials: K
authors.lastname: Zimmermann
authors.forename: P August
authors.initials: PA
authors.lastname: Schubiger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics*; D002460:Cell Line; D002614:Chelating Agents / Q000737:chemistry; D004353:Drug Evaluation, Preclinical; D005260:Female; D006031:Glycosylation; D006573:Heterocyclic Compounds, 1-Ring / Q000493:pharmacokinetics*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008187:Lutetium / Q000493:pharmacokinetics*; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D039842:Neural Cell Adhesion Molecule L1 / Q000276:immunology*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D011868:Radioisotopes / Q000493:pharmacokinetics*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D006573:Heterocyclic Compounds, 1-Ring; D039842:Neural Cell Adhesion Molecule L1; D011868:Radioisotopes; D019275:Radiopharmaceuticals; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D008187:Lutetium