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 (Ga-67) 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 Ga-67-SDF with nonspecific human IgG was performed by reacting freshly thiolated IgG with the reaction product of MESS and DFO, followed by Ga-67 labeling of the resulting conjugate using GaCl3 (Ga-67-DFO-MESS-IgG). For comparison, Ga-67-DFO conjugated nonspecific human IgG with a nonmetabolizable linkage was synthesized under the same conjugation conditions as those for Ga-67-DFO-MESS-IgG, using a nonmetabolizable heterogeneous bifunctional reagent [N-[(6-maleimidocaproyl)oxy]succinimide, EMCS] instead of MESS (Ga-67-DFO-EMCS-IgG). HPLC size-exclusion chromatography of both preparations showed a single radioactivity and UV peak corresponding to the intact IgG. Generation of Ga-67-SDF from the Ga-67-DFO-MESS-IgG was demonstrated by reverse-phase HPLC analysis and cellulose acetate electrophoresis after the incubation of Ga-67-DFO-MESS-IgG in a buffered solution containing carboxyesterase. After injection of Ga-67-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 Ga-67-DFO-EMCS-IgG was injected. These results indicate that MESS has the ability to conjugate antibodies with Ga-67-SDF via ester bonding and to release Ga-67-SDF by the action of carboxyesterase both in vitro and in vivo.