title: Cytotoxicity of glucose oxidase conjugated with antibodies to target cells: killing efficiency depends on the conjugate internalization.
abstract: The cytotoxic action of glucose oxidase conjugated with antibodies against the target cells has been examined in a culture of human endothelial cells. Internalizable (anti-endothelial, MoAb E25) and non-internalizable (anti-fibronectin, MoAb FN) monoclonal antibodies were employed as vectors. Anti-endothelial monoclonal antibody E78 (whether it can be internalized by endothelial cells is unclear) and polyclonal mouse antiserum to the human endothelium were also used. The conjugates were prepared by oxidation of the enzyme carbohydrate moiety with periodate. Free conjugates display similar enzyme activity in glucose solution. In contrast to glucose oxidase, conjugated with no-immune IgG, antibody-conjugated glucose oxidase binds specifically to target cells. The efficiency of targeting was different for various conjugates. Targeting via the anti-fibronectin antibody and anti-endothelial antiserum provided maximal quantitative binding of glucose oxidase to endothelial cells, while the conjugates with MoAb E25 and MoAb E78 monoclonal antibodies provided less effective binding. In the presence of glucose, targeted glucose oxidase generated H2O2. Hydrogen peroxide is relatively stable in buffer, but rapidly decays in the culture medium supplemented with 20% human serum. Though the quantitative binding of MoAb E25-conjugated glucose oxidase was minimal comparing to other conjugates, targeting via MoAb E25 produced the maximal cytotoxic effect as well as targeting via polyclonal antiserum. The killing efficiencies of MoAb FN-conjugated and MoAb E78-conjugated glucose oxidase were about 30-fold lower. The high efficiency of the MoAb E25-conjugated enzyme may be due to its internalization by target cells. Internalization can lead to unaccessibility of generated H2O2 for extracellular scavengers and pH optimization for glucose oxidase activity, which provides valuable advantages for the cytotoxicity of the conjugate. Thus, cytotoxicity of antibody-conjugated glucose oxidase depends not only on the efficiency of specific binding to the target cell, but also on the fate of cell-bound conjugate. Cytotoxicity is extremely effective in case of 'internalizable' conjugate and drastically less effective in case of 'non-internalizable' conjugate.
authors.affiliation: Institute of Experimental Cardiology, National Cardiology Research Center, Academy of Medical Sciences, Moscow, U.S.S.R.
authors.forename: V R
authors.initials: VR
authors.lastname: Muzykantov
authors.forename: O V
authors.initials: OV
authors.lastname: Trubetskaya
authors.forename: E A
authors.initials: EA
authors.lastname: Puchnina
authors.forename: D V
authors.initials: DV
authors.lastname: Sakharov
authors.forename: S P
authors.initials: SP
authors.lastname: Domogatsky
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002470:Cell Survival; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology; D005353:Fibronectins / Q000276:immunology; D005949:Glucose Oxidase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D000922:Immunotoxins / Q000494:pharmacology*; D051379:Mice; D014471:Umbilical Veins
chemical_list: D000911:Antibodies, Monoclonal; D005353:Fibronectins; D000922:Immunotoxins; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase