Disulfide bond-targeted radiolabeling: Tumor specificity of a streptavidin-biotinylated monoclonal antibody complex

A site-specific labeling method was developed in which sulfhydryl groups of a murine IgG2a anti-ovarian monoclonal antibody, 5G6.4, were biotinylated with N-iodoacetyl-N′-biotinylhexylenediamine (Compound 1) following partial reduction of disulfide bonds with dithiothreitol. Reaction of 1-alkylated 5G6.4 with ¹²⁵I-streptavidin gave immunoreactive streptavidin-1-biotinylated complexes. Radio-fast protein liquid chromatography data were consistent with the formation of a stable monovalent streptavidin-half-antibody complex as the major species. In vivo specific localization of these radioantibody conjugates to human tumor xenografts of ovarian carcinoma was confirmed by a comparative biodistribution study in nude mice using as a control the nonspecific ¹²⁵I-streptavidin-1-alkylated UPC-10 (an irrelevant IgG2a monoclonal antibody) complex prepared analogously as described above. Tumor uptake for radiolabeled 5G6.4 [0.279±0.041 % (SE) kg injection dose/g) was significantly greater [P < 0.025] than for UPC-10 [0.165±0.027% kg injection dose/g]. The tumor:blood ratio (738±1.285) for 5G6.4 was ≈3 times that for UPC-10 (2.48 ± 0.708, P < 0.01). This sulfhydryl site-directed approach demonstrated that reduced disulfides of monoclonal antibodies are viable sites for attaching labels without significant loss of in vitro and in vivo immunoreactivity.