Immunoconjugates joined by thioether bonds having reduced toxicity and improved selectivity

DETAILED DESCRIPTION I claim: 1.
A method for producing a 1:1 protein:anitbody tioether-linked immunoconjugate as a predominant species, comprising the steps of: combining a protein selected from the group consisting of toxin, carrier protein, biological response modifier, chelating protein or chelating peptide, with a heterobifunctional reagent having the following general formula: ##STR7## wherein R.
sub.
1 is: ##STR8## wherein n=1 to 10; and wherein R.
sub.
2 is selected from the group consisting of o- and p-nitrophenyl, 2-chloro-4-nitrophenyl, cyanomethyl, 2-mercaptopyridyl, hydroxybenztriazole, N-hydroxysuccinimide, trichlorophenyl, 2-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 2,3,5,6,-tetrafluorophenyl, tetrafluorothiophenyl, o-nitro-p-sulfophenyl, N-hydroxyphthalimide, N,N-diethylamino, N-hydroxypyroolidone, under conditions that permit reaction of the protein and R.
sub.
2, thereby forming a derivatized protein; separating the derivatized protein; reacting an antibody or antibody fragment with a reducing agent, thereby forming a reduced antibody component; separating the reduced antibody component; and mixing the derivatized protein and the reduced antibody component in a molar ration of approximately 0.
5:1 to approximately 5:1 at room temperature for about 5 minutes to about 60 minutes, such that at least one thiol group of the reduced antibody component and at least one maleimide group of the derivatized protein combine to form a thioether bond, thereby producing the 1:1 protein:antibody thioether-linked immunoconjugate as a predominant species.
2.
The method of claim 1 wherein the toxin is selected from the group consisting of Pseudomonas exotoxin A, ricin, abrin and diphtheria toxin.
3.
The method of claim 1 wherein the heterobifunctional reagent is succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) or derivatives thereof.
4.
The method of claim 1 wherein the antibody fragment is F(ab').
sub.
2 fragment of F(ab') fragment.
5.
The method of claim 1 wherein the antibody is genetically engineered antibody or fragments thereof having an available sulfhydryl group