A novel bifunctional metabolizable linker for the conjugation of antibodies with radionuclides

A novel heterogeneous bifunctional reagent containing an ester bond, N-[[4-(2-maleimidoethoxy)-succinyl]oxy]succinimide (MESS), was designed and synthesized for the conjugation of antibodies with the gallium-67 (67Ga) chelate of succinyldeferoxamine (SDF) via the ester bond. MESS was synthesized by the acylation of N-(2-hydroxyethyl)maleimide with succinic anhydride, followed by the activation of the resulting carboxylic acid to a succinimido ester. MESS possesses a maleimide group for protein conjugation and an active ester group for deferoxamine (DFO) coupling, and the two functional groups are linked via ester bonding. Conjugation of 67Ga-SDF with nonspecific human IgG was performed by reacting freshly thiolated IgG with the reaction product of MESS and DFO, followed by 67Ga labeling of the resulting conjugate using GaCl3 (67Ga-DFO-MESS-IgG). For comparison, 67Ga-DFO conjugated nonspecific human IgG with a nonmetabolizable linkage was synthesized under the same conjugation conditions as those for 67Ga-DFO-MESS-IgG, using a nonmetabolizable heterogenous bifunctional reagent [N-[(6-maleimidocaproyl)oxy]succinimide, EMCS] instead of MESS (67Ga-DFO-EMCS-IgG). HPLC size-exclusion chromatography of both preparations showed a single radioactivity and UV peak corresponding to the intact IgG. Generation of 67Ga-SDF from the 67Ga-DFO-MESS-IgG was demonstrated by reverse-phase HPLC analysis and cellulose acetate electrophoresis after the incubation of 67Ga-DFO-MESS-IgG in a buffered solution containing carboxyesterase. After injection of 67Ga-DFO-MESS-IgG into mice, faster radioactivity clearance from the blood and less radioactivity accumulation in the liver, kidney, and spleen was noted than when 67Ga-DFO-EMCS-IgG was injected