Astatine-211 labeling of an antimelanoma antibody, NR-ML-05, and its Fab fragment with N-succinimidyl p-[211At]astatobenzoate (2a) has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 in the reaction mixture. The dry distillation technique employed gave recoveries of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [211At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[125I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

At 100 °C (Reflux Temperature). A solution of 104 mg (0.414 mmol) of 34 dissolved in 7 mL of 95% formic acid was heated at reflux under nitrogen for 12 h. The formic acid was removed with a rotary evaporator, and the residue was diluted with 50 mL of 10% aqueous NaHCOs. The aqueous solution was extracted with 100 mL of CH2C12, and the organic phase was washed with brine, dried over Na2S04, and concentrated to give 112 mg of a yellow oil. The crude product was purified by flash chromatography on 5 g of silica using a solvent gradient ranging from 1:4 to 1:1 EtOAc-hexane. The less polar fractions gave 23.3 mg (24%) of 38. The more polar fractions yielded 63.2 mg (55%) of (lfiS,4SR,7fiS,8fiS,10Sk)-2-aza-8-(formyloxy)-7-methyl-10- (1) Reported in part in the thesis of Bacon, E.

Protein sulfhydryl reactive N-(4-[125I]iodophenethyl)maleimide (IPEM, 5) was obtained from N-[4-(tri-n-butylstannyl)phenethyl]maleimide in 59-100% radiochemical yield. Conjugation of 5 to NR-ML-05 Fab, a murine anti-melanoma antibody Fab fragment that had been previously reduced with dithiothreitol (DTT), was effected in an average of 85% yield. Results from in vitro chemical challenges and serum stability studies on the IPEM conjugate of NR-ML-05 Fab (6) indicated a stable covalent attachment of the radioiodine. A biodistribution study of the IPEM conjugate in tumor-bearing athymic nude mice showed lack of significant accumulation of radioiodine in the thyroid and stomach which was an indication of in vivo stability. The observed uptake in tumor was consistent with that obtained for Chloramine-T- or p-iodobenzoate-labeled NR-ML-05 Fab conjugates.

The development of monoclonal antibodies of high affinity and selectivity for tumor antigens has supported the development of radiolabeled antibodies for diagnostic localization and targeted delivery of therapeutic radionuclides. Several radionuclide chelating agent systems have been developed for indium-111 and technetium-99m that have shown good sensitivity and specificity for tumor detection in patients. Feasibility for therapy has been shown in animal models and a few patient studies with iodine-131 and yttrium-90. This review covers selection of radionuclides and chemistry of antibody radiolabeling.

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was approximately a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate.