Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (beta G-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-beta G-sPEG conjugate showed that mAb 1E8 (IgG1) bound beta G and beta G-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the beta G activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared beta G-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared beta G-sPEG more completely and rapidly than 1E8, reducing the serum concentration of beta G-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-beta G-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of beta G-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-beta G-PEG conjugates for glucuronide prodrug therapy of cancer.