Site-pecific ^99mTc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain

The development of an antibody labeling method with ^99mTc is important for cancer imaging. Most bifunctional chelate methods for ^99mTc labeling of antibody incorporate a ^99mTc chelator through a linkage to lysine residue. In the present study, a novel site-specific ^99mTc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to produce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with ^99mTc. The number of conjugated DHZ was 1.7 per antibody. ^99mTc labeling efficiency was 46-85% for T101 and 67-87% for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T101 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with ^99mTc. Moreover, ^99mTc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.