COBRA VENOM FACTOR IMMUNOCONJUGATES - EFFECTS OF CARBOHYDRATE-DIRECTED VERSUS AMINO GROUP-DIRECTED CONJUGATION

Human IgM monoclonal antibody 16-88, derived from patients immunized w ith autologous colon carcinoma cells, was derivatized with two differe nt cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2-pyridyldithio)propi onate (SPDP), which is amino group-directed. Two antibody functions, a ntigen binding and complement activation, were assayed upon derivatiza tion with TPCH and SPDP. TPCH allowed for extensive modification (up t o 17 TPCH molecules per antibody) without impairment of antigen bindin g activity, while this function was significantly compromised upon der ivatization with SPDP. Antibody molecules derivatized with 16 SPDP res idues showed almost complete loss of their antigen binding function. T he complement activating ability of antibody 16-88 was significantly d ecreased after derivatization with TPCH or SPDP. In the case of SPDP d erivatization, this decrease of the complement activating ability is p redominantly a consequence of the impaired binding function. Upon conj ugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein w hich is capable of activating the alternative pathway of complement, t he antigen binding activity of SPDP-derivatized antibody was further c ompromised, whereas that of TPCH-derivatized antibody remained unaffec ted even after attachment of three or four CVF molecules per antibody. In both conjugates CVP retained good functional activity. CVF was sli ghtly more active when attached to SPDP-derivatized antibody, suggesti ng a better accessibility of amino group-coupled CVF for its interacti on with other complement proteins. These results indicate that carbohy drate-directed conjugation compromises the antibody function of comple ment activation, but allows for the generation of immunoconjugates wit h unimpaired antigen binding capability. Accordingly, carbohydrate-dir ected cross-linkers may contribute to improve the efficacy of immunoco njugates in cancer therapy.