Radiochemistry

²²⁵Ac (ORNL, Oak Ridge, TN) was conjugated to the hu11B6 antibody or isotype-matched control antibody and purified using a 2-step labeling procedure^{4, 20}. Activity was measured at secular equilibrium with a Squibb CRC-17 Radioisotope Calibrator (E.R. Squibb and Sons, Inc., Princeton, NJ) set at 775 and multiplying the displayed activity value by 5. The radiochemical purity of the final product, [²²⁵Ac]hu11B6, was determined using instant thin-layer chromatography (ITLC) with a stationary phase of silica gel impregnated paper (Gelman Science Inc., Ann Arbor, MI) and two different mobile phases. Mobile phase I is 10 mM ethylenediaminetetraacetic acid and II is 9% sodium chloride/10 mM sodium hydroxide. The strips were counted in a Packard Cobra γ-counter (Packard Instrument Co., Inc., Meriden, CT) using a 370–510 KeV window. The purified radioimmunoconstruct was formulated in a solution of 1% human serum albumin (HSA, Swiss Red Cross, Bern, Switzerland) and 0.9% sodium chloride (Normal Saline Solution, Abbott Laboratories, North Chicago, IL) for intravenous injection. Radiochemically pure ²²³Ra was eluted from an Actinium-227 source, as described²⁷ and formulated in 0.03 M citrate in saline. An empirically derived calibrator setting of #277, using a CRC-127R dose calibrator (Capintec Inc), was used to dose ²²³Ra.

⁸⁹Zr (MSKCC Radiochemistry and Imaging Probes (RMIP) Core Facility or 3D Imaging, Little Rock AR) was conjugated to a hu11B6-DFO antibody as previously described^{10, 24}. Activity was measured with a Squibb CRC-17 Radioisotope Calibrator set at #465. Briefly, 8.5 mCi of ⁸⁹Zr[Zr]oxalate was neutralized with 0.025 mL of 1 M Na₂CO₃ to pH 7.0. Any colloidal precipitate was removed with centrifugal filtration (0.22 µm Corning Spin-X filter at 1000×g) and 0.25 mL of 11B6-DFO (8.8 g/L; Fuji Film; lot # NBS0131-6-2 (Prost B3298) in 25 mM sodium acetate buffer (pH 5.5) was added to the clear neutralized ⁸⁹Zr[Zr] filtrate. The reaction mixture was purified after 75 min at ambient temperature by size exclusion chromatography using a 10DG column (BioRad) mobile phase and a 1% HSA mobile phase. The radiochemical purity of the final product, [⁸⁹Zr]hu11B6, was assayed using ITLC (silica gel impregnated paper and Mobile Phases I and II) as described above.