title: Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.
abstract: Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy.
  
  
  METHODS
  The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26.
  
  
  RESULTS
  At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo.
  
  
  CONCLUSIONS
  According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.
authors.affiliation: Department of Nuclear Medicine, University of Heidelberg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Eisenhut
authors.forename: W D
authors.initials: WD
authors.lastname: Lehmann
authors.forename: W
authors.initials: W
authors.lastname: Becker
authors.forename: T
authors.initials: T
authors.lastname: Behr
authors.forename: H
authors.initials: H
authors.lastname: Elser
authors.forename: W
authors.initials: W
authors.lastname: Strittmatter
authors.forename: A
authors.initials: A
authors.lastname: Steinsträsser
authors.forename: R P
authors.initials: RP
authors.lastname: Baum
authors.forename: T
authors.initials: T
authors.lastname: Valerius
authors.forename: R
authors.initials: R
authors.lastname: Repp
authors.forename: Y
authors.initials: Y
authors.lastname: Deo
mesh_terms: D000818:Animals; D006801:Humans; D007553:Isotope Labeling; D008624:Mercaptoethylamines / Q000138:chemical synthesis / Q000493:pharmacokinetics; D016719:Radioimmunodetection; D051381:Rats; D017207:Rats, Sprague-Dawley; D011933:Reagent Kits, Diagnostic; D013388:Succinimides / Q000138:chemical synthesis / Q000493:pharmacokinetics; D013667:Technetium; D014018:Tissue Distribution
chemical_list: D008624:Mercaptoethylamines; D011933:Reagent Kits, Diagnostic; D013388:Succinimides; C099928:6-(4'-(4''-carboxyphenoxy)butyl)-2,10-dimercapto-2,10-dimethyl-4,8-diazaundecane; D013667:Technetium