Tumor imaging with technetium-99m-labeled hydrazinonicotinamide-Fab' conjugates.

UNLABELLED: This study compares the in vivo properties of direct versus indirect 99mTc-labeling for two Fab' fragments from antibodies that recognize tumor-associated antigens. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Preparation and preliminary evaluation of technetium-99m-labeled fragment E1 for thrombus imaging.

Fragment E1 labeled with 123I has been previously shown to permit imaging of thrombi in patients within as little as 20 min after injection. Because of the relatively rapid localization and blood disappearance of this protein, 99mTc would be the most clinically acceptable radionuclide for labeling Fragment E1. In this study, human fragment E1 was derivatized with a hydrazino nicotinate function to permit radiolabeling with reduced technetium. The modification reaction was carried out while the fragment E1 was protected in a complex, so that the modification occurred in nonfunctional regions of the fragment E1 molecule. After radiolabeling with 99mTc, the modified fragment E1 retained its functional activity, as judged by its binding to fragment DD in vitro. The ability of 99mTc-fragment E1 to produce images of venous thrombi was demonstrated in animal models. Images were focally positive within 20 min to 1 hr after injection. Thrombus-to-blood ratios exceeded those from 125I-fibrinogen in the same animals. This method of labeling appears to provide an alternative radiolabel to 123I without compromising the function of fragment E1.

Preparation of hydrazino-modified proteins and their use for the synthesis of 99mTc-protein conjugates.

The syntheses and protein linking properties of succinimidyl 4-hydrazinobenzoate hydrochloride (SHBH) and succinimidyl 6-hydrazinonicotinate hydrochloride (SHNH), two new heterobifunctional linkers which lead to hydrazino-modified proteins, are described. SHBH-modified proteins are unstable due to the presence of the phenylhydrazine moiety. This problem was overcome by synthesizing the hydrazinopyridine analogue SHNH, and the conjugates derived from this linker are stable. Tc(V) oxo precursors readily add to hydrazinopyridine-modified proteins to yield the desired 99mTc-radiolabeled protein. 99mTc-hydrazinopyridine-polyclonal IgG conjugates are useful agents for the imaging of focal sites of infection.

Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats.

The biologic behavior of human polyclonal immunoglobulin (IgG) radiolabeled with technetium-99m (99mTc) by a novel method, via a nicotinyl hydrazine derivative, was evaluated in rats. Technetium-99m- and indium-111-IgG were co-administered to normal rats and biodistribution was determined at 2, 6, and 16 hr. The inflammation imaging properties of the two reagents were compared in rats with deep-thigh infection due to Escherichia coli. Blood clearance of both antibody preparations was well described by a bi-exponential function: (99mTc-IgG: t1/2 = 3.82 +/- 0.89 and 57.52 +/- 1.70 hr. 111In-IgG: 3.93 +/- 0.117 and 40.71 +/- 1.26 hr). Biodistributions in the solid organs were similar, however, small but statistically significant differences were detected: 99mTc-IgG greater than 111In-IgG in lung, liver, and spleen; 99mTc-IgG less than 111In-IgG in kidney and skeletal muscle (p less than 0.01). At all three imaging times, target-to-background ratio and percent residual activity for the two compounds were remarkably similar. These studies establish that human polyclonal IgG labeled with 99mTc via a nicotinyl hydrazine modified intermediate is equivalent to 111In-IgG for imaging focal sites of infection in experimental animals.