IMMUNOCONJUGATES JOINED BY THIOETHER BONDS HAVING REDUCED TOXICITY AND IMPROVED SELECTIVITY

Method for producing an immunoconjugate comprising the steps of reacting a toxin or protein with a heterobifunc-tional reagent having the following general formula: where R1 is: -(CH2)n-or or where n = 1 to 10; and where R2 is selected from the group consisting of o- and p-nitrophenyl, 2-chloro-4-nitrophenyl, cyanomethyl, 2-mercapto-pyridyl, hydroxybenztriazole, N-hydroxysuccinimide, trichloro-phenyl, tetrafluorophenyl, 2-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, o-nitro-p-sulfophenyl, N-hydroxyphthali-mide, N,N-diethylamino, N-hydroxypyrrolidone, tetrafluorothio-phenyl, and 2,3,5,6-tetrafluorophenyl, under reactive condi-tions, thereby forming a derivatized toxin or protein. The derivatized toxin or protein is separated from the reaction mixture and combined with an antibody or antibody fragment under reactive conditions, such that at least one native disulfide bond is reduced to produce at least one thiol group, capable of forming a thioether bond between said thiol group and the maleimide group of said derivatized toxin, or protein thereby forming an immunoconjugate. The preferred heterobifunc-tional linking group is succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC).