Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

[Oxidative inactivation of angiotensin-converting enzyme]. / Oksidativnaia inaktivatsiia angiotenzinprevrashchaiushchego fermenta.

Hydrogen peroxide inactivates the purified human angiotensin-converting enzyme (ACE) in vitro; the inactivating effect of H2O2 is eliminated by an addition of catalase. The lung and kidney ACE are equally sensitive to the effect of hydrogen peroxide. After addition of oxidants (H2O2 alone or H2O2 + ascorbate or H2O2 + Fe2+ mixtures) to the membranes or homogenates of the lung, the inactivation of membrane-bound ACE is far less pronounced despite the large-scale accumulation of lipid peroxidation products. The marked inactivation of ACE in the membrane fraction (up to 55% of original activity) was observed during ACE incubation with a glucose:glucose oxidase:Fe2+ mixture. Presumably the oxidative potential of H2O2 in tissues in consumed, predominantly, for the oxidation of other components of the membrane (e.g., lipids) rather than for ACE inactivation.

Cytotoxicity of glucose oxidase conjugated with antibodies to target cells: killing efficiency depends on the conjugate internalization.

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.

In vivo administration of glucose oxidase conjugated with monoclonal antibodies to angiotensin-converting enzyme. The tissue distribution, blood clearance, and targeting into rat lungs.

A conjugate between glucose oxidase (GO) and monoclonal antibody to human angiotensin-converting enzyme (ACE) cross-reacting with rat ACE (MoAb9b9) has been prepared by oxidation of the cardohydrate moiety of the enzyme with sodium periodate. The conjugate (GO-MoAb9b9) thus obtained retained both antigen-binding capacity and enzymatic activity. The fate of the conjugate in vivo after intravenous injection was studied using conjugates containing radiolabeled enzyme. GO-MoAb9b9 was specifically accumulated in rat lungs upon in vivo administration, as compared with free enzyme and nonimmune IgG-conjugated glucose oxidase. The specificity of the conjugate accumulation expressed as the localization ratio (the ratio between radioactivity of gram tissue to that of blood) (Loc. Ratio) reached a value up to 50 on the second day after injection, in contrast to native enzyme and to IgG-conjugated enzyme (Loc. Ratio was less than 0.5 for both preparations). The Loc. Ratio of GO-MoAb9b9 was even higher than that of the original antibody MoAb9b9 and was equal to 20, which is probably explained by an extremely rapid blood clearance of the conjugate from the circulation. The administration of excess free MoAb9b9 dramatically inhibited the conjugate targeting in the lung without any effect on liver uptake. At doses ranging from 10 to 1,000 micrograms/rat, the conjugate was accumulated in the lung without saturation of the antigen determinants of the target. At minimal doses, the efficiency of targeting achieved 5 to 7% of the conjugate injected. With elevation of the dose, the efficiency of targeting decreased to 2.5% of the injected dose (1 mg of GO-MoAb9b9 per rat). A sixfold greater accumulation of unmodified radiolabeled MoAb9b9 compared with the GO-MoAb9b9 conjugate in rat lung has been observed, though the kinetics of desorption from the target organ was similar for both the antibody and the conjugate. In the bloodstream, the conjugate persisted for at least 5 days without binding to blood cells; all circulating radioactivity was associated with proteins. A considerable part of the conjugate (to 50%) circulated as a tight antibody-enzyme complex for several days. The conjugate retained its antigen-binding capacity for at least 24 h; during this period, its enzymatic activity decreased by less than 40%. The results obtained provide the experimental ground for further attempts to apply glucose oxidase conjugates for local modulation of inflammation and elimination of the target cells.