Immunological and biological stability of immunotoxins in vivo as studied by the clearance of disulfide-linked pokeweed antiviral protein-antibody conjugates from blood.

Monoclonal antibodies against human T-cell antigen 3A1, human transferrin receptor, and mouse Thy 1.1 antigen were linked to pokeweed antiviral protein (PAP) by a disulfide bond. Because the ability of the immunotoxin to home on target cells in vivo and the eventual internalization of the hemitoxin polypeptide depends in part on the stability of the conjugate in circulation, the clearance of antibody-PAP conjugates from blood was investigated. Blood samples collected from rabbits at different times after the injection of immunotoxin were analyzed for: (a) total mouse IgG; and (b) intact antibody-PAP conjugate in enzyme-linked immunosorbent assay. Further, antibody-PAP conjugate was separated from PAP by differential precipitation using polyethyleneglycol, and the PAP content of the fractions were analyzed by radioimmunoassay. Free PAP is removed very rapidly from blood, and 95% is cleared within 2 h. Our results showed that the immunotoxin did not dissociate in circulation immediately, and about 90% of the initial concentration of the conjugate was still present for more than 4 h. Analysis by enzyme-linked immunosorbent assay showed a 4- to 8-h lag period in which immunotoxin concentrations were relatively unchanged. This was followed by a steady decline, and the half-life of the conjugate in circulation then ranged between 17 and 24 h. Not only did the immunotoxins remain intact immunologically, but they also retained their biological activity as measured by the ability of blood-borne immunotoxins to efficiently block protein synthesis of target cells in vitro. These data show that the disulfide linkage of toxin to antibody is reasonably stable and that the immunotoxin retains the biological properties of both the antibody and the hemitoxin polypeptide in circulation.