Renal metabolism of 3 '-iodohippuryl N-epsilon-maleoyl-L-lysine (HML)-conjugated Fab fragments

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3'-[I-131] iodohippuryl N-is an element of-maleoy l-L-lysine (HML), that liberates m-iodohippuric acid before antibody fragme nts are incorporated into renal cells. In mice, HML-conjugated Fab demonstr ated low renal radioactivity levels from early postinjection times. In this study, renal metabolism of HML-conjugated Fab fragments prepared by differ ent thiolation chemistries and by direct radioiodination were investigated to determine the mechanisms responsible for the low renal radioactivity lev els. Fab fragments were thiolated by 2-iminothiolane modification or by red uction of disulfide bonds in the Fab fragments, followed by conjugation wit h radioiodinated HML to prepare [I-131]HML-IT-Fab and [I-125]HML-Fab, respe ctively. In biodistribution studies in mice, both [I-131]HML-IT-Fab and [I- 125]HML-Fab demonstrated significantly lower renal radioactivity levels tha n those of [125I]Fab. In subcellular distribution studies, [I-125]Fab showe d migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection. On the other hand, the ma jority of the radioactivity was detected only in the membrane fraction at t he same time points after injection of both [I-131]HML-IT-Fab and [I-125]- HML-Fab. In metabolic studies, while [I-125]Fab remained intact at 10 min p ostinjection, both HML-conjugated Fab fragments generated m-iodohippuric ac id as a radiometabolite at the same postinjection time. [I-131]HML-IT-Fab r egistered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [I-125]HML-Fa b. This suggested that metabolism of both HML-conjugated Fab fragments woul d occur in the membrane fractions of the renal cells. The findings of this study reinforced our previous hypothesis that radiochemical design of antib ody fragments that liberate radiometabolites that are excreted into the uri ne by the action of brush border enzymes would constitute a useful strategy to reduce renal radioactivity levels from early postinjection times.