Conjugation of antibodies with DOTA-NHS and radiolabeling with indium-111 or lutetium-177

Typically, 2 mg of Ts29.2 or 16F12 antibodies (29 μL, 42 mg/mL in PBS) were added to 2 mg of DOTA-NHS ester freshly introduced in a low retention tube (ratio DOTA-NHS/antibody: 200/1). The resulting solution was diluted with DPBS buffer (600 μL) and the pH of the reaction mixture was adjusted to 7.2-7.4 by portionwise addition of a 0.1 M aqueous sodium hydroxide solution (100-130 μL). The resulting mixture was stirred (end-over-end rotation) overnight at 4 °C. After return back to room temperature, the reaction mixture was transferred on an Amicon® Ultra centrifugal filter (50K, Millipore) and the tube was rinsed twice with 1 mL of Milli Q water. The resulting diluted reaction mixture was concentrated by centrifugation to less than 100 μL and purified by semi-preparative size exclusion HPLC. The fractions containing the DOTA-Ts29.2 or DOTA-16F12 conjugate were collected (3-4 mL; R_t: 20.1 min for DOTA-Ts29.2; R_t: 19.8 min for DOTA-16F12) and concentrated to less than 100 μL by centrifugation using an Amicon® Ultra centrifugal filter (50K, Millipore). The final concentration was estimated using Nanodrop and the number of grafted DOTA per antibody was determined using MALDI-TOF mass spectrometry.

For radiolabeling, 2 μL of the previously concentrated solution of DOTA-Ts29.2 or DOTA-16F12 conjugates (22.7 μg/μL) were diluted with 0.1 M HEPES buffer (pH 5.5, 248 μL). Then, a solution of [¹¹¹In]InCl₃ or [¹⁷⁷Lu]LuCl₃ in 0.05 M HCl (60 μL, 17 MBq) was added and the reaction mixture was vortexed before incubation at 45 °C for 15-20 min. The radiochemical purities of resulting [¹¹¹In]DOTA-Ts29.2, [¹⁷⁷Lu]DOTA-Ts29.2 or [¹⁷⁷Lu]DOTA-16F12 were determined using size exclusion analytical radio-HPLC analyses.