Derivatization of cetuximab and F(ab′)2 fragments for radiolabeling

Conjugation was performed at a 20:1 DOTAGA-anhydride/cetuximab molar ratio. 100 μL of a 3.7 mg.mL^− 1 suspension of DOTAGA-anhydride (370 μg, 0.8 μmol, 20 equiv) in dry chloroform (Carlo Erba, Val de Reuil, France) were pipetted under ultrasonication and transferred into a 15 mL polypropylene tube. The chloroform was evaporated under a gentle stream of air. 480 μL of a solution of purified cetuximab (12.5 mg.mL^− 1, 6 mg, 40 nmol, 1 equiv) in PBS, pH 7.4, (Fisher Scientific, Illkirch, France) were subsequently added. The solution was completed to 3 mL with PBS 0.1 M, pH 7.4, and gently mixed at 25 °C for 30 min. Unbound DOTAGA was then removed by ultrafiltration (Vivaspin filter 10 kDa, Sartorius, 30 min at 1520 g, 4 °C). Conjugated cetuximab was washed twice with 5 mL of PBS 0.1 M, pH 7.4, and the concentrated solution was diluted in 500 μL of ammonium acetate buffer 0.1 M pH 5.9. The purified immunoconjugate DOTAGA-cetuximab was stored at 4 °C. Concentration of the antibody was determined by UV spectrophotometry at 280 nm (E₂₈₀ = 1.44 mg mL^− 1 cm^− 1). The degree of labeling was determined by MALDI-TOF mass spectrometry using sinapinic acid as matrix.

Following a similar procedure, conjugation was performed at a 15:1 DOTAGA-anhydride/F(ab′)₂ molar ratio. 111 μL of a 3.7 mg.mL^− 1 suspension of DOTAGA-anhydride (411 μg, 0.9 μmol, 15 equiv) in dry chloroform were pipetted under ultrasonication and transferred into a 15 mL polypropylene tube. See details in Supplemental methods.