title: Cobra venom factor immunoconjugates: effects of carbohydrate-directed versus amino group-directed conjugation.
abstract: Human IgM monoclonal antibody 16-88, derived from patients immunized with autologous colon carcinoma cells, was derivatized with two different cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is amino group-directed. Two antibody functions, antigen binding and complement activation, were assayed upon derivatization with TPCH and SPDP. TPCH allowed for extensive modification (up to 17 TPCH molecules per antibody) without impairment of antigen binding activity, while this function was significantly compromised upon derivatization with SPDP. Antibody molecules derivatized with 16 SPDP residues showed almost complete loss of their antigen binding function. The complement activating ability of antibody 16-88 was significantly decreased after derivatization with TPCH or SPDP. In the case of SPDP derivatization, this decrease of the complement activating ability is predominantly a consequence of the impaired binding function. Upon conjugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein which is capable of activating the alternative pathway of complement, the antigen binding activity of SPDP-derivatized antibody was further compromised, whereas that of TPCH-derivatized antibody remained unaffected even after attachment of three or four CVF molecules per antibody. In both conjugates CVF retained good functional activity. CVF was slightly more active when attached to SPDP-derivatized antibody, suggesting a better accessibility of amino group-coupled CVF for its interaction with other complement proteins. These results indicate that carbohydrate-directed conjugation compromises the antibody function of complement activation, but allows for the generation of immunoconjugates with unimpaired antigen binding capability.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Georgetown University, Washington, D.C. 20007, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zara
authors.forename: N
authors.initials: N
authors.lastname: Pomato
authors.forename: R P
authors.initials: RP
authors.lastname: McCabe
authors.forename: R
authors.initials: R
authors.lastname: Bredehorst
authors.forename: C W
authors.initials: CW
authors.lastname: Vogel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D003110:Colonic Neoplasms / Q000276:immunology; D003167:Complement Activation / Q000187:drug effects*; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000031:analogs & derivatives; D004546:Elapid Venoms / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D006168:Guinea Pigs; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D007075:Immunoglobulin M; D000922:Immunotoxins / Q000494:pharmacology; D011725:Pyridines; D013388:Succinimides
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003432:Cross-Linking Reagents; D004546:Elapid Venoms; D007075:Immunoglobulin M; D000922:Immunotoxins; D011725:Pyridines; D013388:Succinimides; C016310:cobra venom factor; C070209:S-(2-thiopyridyl)cysteine hydrazide; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D003545:Cysteine