Clinical-scale radiolabeling of a humanized anticarcinoembryonic antigen monoclonal antibody, hMN-14, with residualizing 131I for use in radioimmunotherapy.

UNLABELLED

Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of (131)I (residualizing (131)I) 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 (131)I moiety, (131)I-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 mumol per 3.7 GBq of (131)I) at a pH of 7.0-7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive (131)I. 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 (131)I 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 > or =95% of the radioactivity being associated with hMN-14 and with < or =4% aggregation. Similar yields were obtained in a subset of radiolabelings (n = 7) with >3.7 GBq of (131)I. 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 approximately 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 (131)I-IMP-R4 and quenched (131)I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel-level preparation of (131)I-IMP-R4-hMN-14 safe, convenient, and practical.