title: Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents.
abstract: A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody.
authors.affiliation: Medical Products Department, E. I. du Pont de Nemours and Company, North Billerica, Massachusetts 01862.
authors.forename: F A
authors.initials: FA
authors.lastname: Liberatore
authors.forename: R D
authors.initials: RD
authors.lastname: Comeau
authors.forename: J M
authors.initials: JM
authors.lastname: McKearin
authors.forename: D A
authors.initials: DA
authors.lastname: Pearson
authors.forename: B Q
authors.initials: BQ
authors.lastname: Belonga
authors.forename: S J
authors.initials: SJ
authors.lastname: Brocchini
authors.forename: J
authors.initials: J
authors.lastname: Kath
authors.forename: T
authors.initials: T
authors.lastname: Phillips
authors.forename: K
authors.initials: K
authors.lastname: Oswell
authors.forename: R G
authors.initials: RG
authors.lastname: Lawton
mesh_terms: D000098:Acetophenones; D000477:Alkylating Agents; D000818:Animals; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D004797:Enzyme-Linked Immunosorbent Assay; D007202:Indicators and Reagents; D007204:Indium; D051379:Mice; D004369:Pentetic Acid; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D000098:Acetophenones; D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D007204:Indium; D004369:Pentetic Acid