Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.
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.
Eisenhut
M
M
Department of Nuclear Medicine, University of Heidelberg, Germany.
Lehmann
W D
WD
Becker
W
W
Behr
T
T
Elser
H
H
Strittmatter
W
W
Steinsträsser
A
A
Baum
R P
RP
Valerius
T
T
Repp
R
R
Deo
Y
Y
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
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