Assessment of Radiochemical Design of Antibodies Using an Ester Bond as the Metabolizable Linkage: Evaluation of Maleimidoethyl 3-(Tri-n-butylstannyl)hippurate as a Radioiodination Reagent of Antibodies for Diagnostic and Therapeutic Applications

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Abstract
Reduction of radioactivity levels in nontarget tissues such as the liver and kidney constitutes a problem to be resolved in diagnostic and therapeutic applications of radiolabeled monoclonal antibodies (mAbs). A new radioiodination reagent with an ester bond to liberate m-iodohippuric acid from covalently conjugated proteins, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was recently developed. MIH liberated m-iodohippuric acid from galactosylneoglycoalbumin in murine liver, and the radiometabolite was rapidly eliminated from the liver into urine as an intact structure. In this study, intact IgG and Fab fragment of a mAb against osteogenic sarcoma were radioiodinated with MIH to further assess the applicability of MIH to radioimmunoimaging and therapy. For comparison, a mAb radioiodinated with N-succinimidyl iodobenzoate (SIB) and indium-111 (111In)-labeled mAbs with diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) were used. Size-exclusion HPLC analysis and cell binding assays indicated the preservation of both structure and antigen binding affinity of radioiodinated MIH−OST7 (IgG). In biodistribution studies in mice, [125I]MIH−OST7 (IgG) showed faster systemic clearance of radioactivity after 24 h postinjection than did [131I]SIB− and [111In]EMCS-Bz-EDTA−OST7 (IgG). [125I]MIH−OST7 (IgG) also exhibited much lower radioactivity levels in nontarget tissues such as the liver and kidney, with higher radioactivity levels in the blood up to 72 h postinjection when compared with [111In]cDTPA−OST7 (IgG). Radioactivity excreted from the mice was found in the urine as m-iodohippuric acid, following administration of [125I]MIH−OST7 (IgG). In athymic mice bearing osteogenic sarcoma, [131I]MIH−OST7 (IgG) indicated higher tumor-to-nontarget ratios of radioactivity at both 24 and 48 h postinjection than [125I]SIB−OST7 (IgG). Although both radioiodinated OST7s showed similar radioactivity levels in the target at 24 h postinjection, a small but significant decrease in the target radioactivity level was observed with [131I]MIH−OST7 (IgG) at 48 h postinjection. In addition, [131I]MIH−OST7 (Fab) showed very rapid cleavage of the ester bond both in vivo and in vitro. These findings indicated that while MIH may be a useful reagent for radioimmunoimaging using IgG mAb, its application to smaller molecular weight mAbs and radioimmunotherapy would be hindered due to the labile characteristics of the ester bond in plasma. Thus, while the present study reinforced the usefulness of metabolizable linkages for reducing nontarget radioactivity levels, a development of plasma-stable metabolizable linkages is also warranted for radioimmunotherapy and for smaller molecular weight polypeptides.

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