Tumor Imaging with Technetium-99m-Labeled Hydrazinonicotinamide-Fab' Conjugates

This study compares the in vivo properties of direct versus indirect 99mTc-labeling for two Fab' fragments from antibodies that recognize tumor-associated antigens. Fab' fragments of two IgG2a monoclonal antibodies were either radiolabeled directly or via the linker bromoacetyl hydrazinonicotinamide hydrobromide (BAHNH) conjugated site specifically at protein thiols. A thiol assay was used to determine the number of thiols in the Fab' and to monitor their consumption during conjugation with BAHNH. Both preparations were labeled to > 95% incorporation of 99mTc, with the isotope tracking the single 50 kD absorbance peak seen on size-exclusion HPLC. The labeled preparations were tested in tumor-bearing and control mice, with dissections at 4 and 24 hr and gamma scintigraphy of the tumor-bearing mice. The major difference between the two labeled preparations for either antibody fragment was the greater accumulation of isotope in the tumor for the indirectly labeled preparations. This increase ranged from 1.5- and 2.7-fold at 4 hr to 2.6- and 3.2-fold at 24 hr for the two antibodies, respectively. Since blood clearance was similar for the two labeling methods, the higher tumor accumulation with the indirectly labeled fragments resulted in higher tumor to blood ratios. Tumors could be imaged with both antibodies with either type of labeling with greater clarity and sensitivity at the 24 hr time point. While both labeling methods resulted in tumor detection through imaging, the images obtained with the indirectly labeled antibody fragments were more easily visualized due to the combination of higher radioisotope accumulation in the tumor and similar blood clearances compared to the direct labeled fragment.