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Basic Science Investigations |
1 Immunomedics, Inc., Morris Plains, New Jersey
2 Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey
Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of 131I (residualizing 131I) involves radioiodinating a small molecular entity, conjugating it to the mAb, and purification. Column purifications are impractical during procedures involving multi-gigabecquerel levels of radioactivity. The goal of this study was to develop a simple, remote, "1-pot" method of radiolabeling and purification for the scaled-up radioiodination of a humanized anti–carcinoembryonic antigen (CEA) mAb, humanized MN-14 (hMN-14; labetuzumab), with an optimized residualizing 131I moiety, 131I-IMP-R4. IMP-R4 is MCC-Lys(MCC)-Lys(X)-D-Tyr-D-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-diethylenetriaminepentaacetic acid. Methods: An IODO-GEN-based remote labeling system was used. IMP-R4 was radioiodinated (0.13 µmol per 3.7 GBq of 131I) at a pH of 7.0–7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive 131I. The product was purified by stirring for 5 min with a 20% (w/v) suspension of an anion-exchange resin and sterilely filtered into a sealed vial. Human serum albumin was added at a final concentration of 1%–2.5%. Immunoreactivity was determined by mixing with CEA and determining the complexation level by size-exclusion high-pressure liquid chromatography. Two control radiolabelings, either with unreduced hMN-14 or with IMP-R4 omitted, also were performed. Results: In 18 radiolabelings with 131I in the range of 2.04–4.81 GBq (55–130 mCi), yields of 59.9% ± 7.9% (mean ± SD) at specific activities of 200 ± 26 MBq/mg (5.4 ± 0.7 mCi/mg) were obtained, with 
95% of the radioactivity being associated with hMN-14 and with 
4% aggregation. Similar yields were obtained in a subset of radiolabelings (n = 7) with >3.7 GBq of 131I. The immunoreactivities of the preparations were typically >95%. Nonspecific incorporation in the absence of IMP-R4 was 0.5%, whereas that obtained with unreduced IgG was 
8%, possibly because of conjugation of IMP-R4 at lysine sites. The process also removed >99% of the quenching reagent used. Radiolabelings performed with freshly prepared solutions or lyophilized preparations produced similar yields, a result that suggested the option for a single-use kit design. Conclusion: Efficient removal of 131I-IMP-R4 and quenched 131I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel–level preparation of 131I-IMP-R4-hMN-14 safe, convenient, and practical.
Key Words: anti–carcinoembryonic antigen antibody • humanized MN-14 (labetuzumab) • IODO-GEN • 131I • radioimmunotherapy
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