Enzymatic activation and trapping of luminol-substituted peptides and proteins. A possible means of amplifying the cytotoxicity of anti-tumor antibodies.

Abstract

Glutathione and glucose oxidase (EC 1.1.3.4) conjugates containing covalently bound luminol were prepared as prototypes for peptides and proteins with latent, enzyme-activatable chemical reactivity. In the presence of small quantities of activated horseradish peroxidase, conjugated luminol molecules were oxidized to unstable free radicals which reacted rapidly with soluble proteins and cells. These observations are of interest in regard to possible sequential localization reactions in which a few molecules of cell-bound antibody-horseradish peroxidase would be used to catalytically alter and trap many molecules of a second (luminol-substituted) enzyme, toxin, or hapten in the same area, as might be desirable in promoting selective cell destruction.Recent studies have indicated that it is feasible to attach toxins to antitumor antibodies as a means of amplifying selective cytotoxicity (1-4), an idea first proposed by Paul Ehrlich in 1906 (5). We have suggested that it may be preferable to attacfi the antibody to an exogenous enzyme capable of converting a protoxin to a toxin, rather than directly to a toxin (1,6,7). This would permit the activity of the antibody to be catalytically amplified, providing for effective toxicity in situations in which only a limited number of conjugated antibody molecules can be localized on the cell surface. Particularly interesting results have been obtained with conjugates of antibody to glucose oxidase, a hydrogen peroxide generating enzyme with a very high turnover number. When glucose oxidase is attached through specific antibody to a target cell, and appropriate cofactors (glucose, 02, lactoperoxidase, and inorganic iodide) are added, the cell is iodinated and cell death occurs. In the complete enzyme system, with iodide or other protoxins, glucose oxidase conjugates of rabbit and goat antibodies to HeLa, HEp-2, L, human colonic carcinoma cells , and mouse myeloma (MOPC-315) cells have shown toxicity for their homologous cell lines in vitro (1, 7-9). Antibody-enzyme conjugates have also been shown to have marked cytotoxicity for bacteria (10) and parasites (11). In all of these cell systems immunologically specific cytotoxicity has been obtained and the antibody-enzyme conjugate has been more potent than the unconjugated antibody in the presence of excess complement.In order to exploit the full potential of enzyme-mediated cytotoxicity in vivo, it will be necessary to localize as many enzyme molecules as possible in the tumor. One approach would be the utilization of a sequential localization reaction in which a few molecules of antibody-enzyme conjugate, specifically bound in the tumor, would be used to catalytically alter and trap many more molecules of a second enzyme in the same area. This might be accomplished by attaching a chemically unreactive organic substituent on the surface of the second enzyme and converting it to a highly reactive molecule at the level of the tumor surface. A theoretical scheme is shown in Fig. 1; El-Ab is antibody-first enzyme conjugate;...