Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (beta G) was examined as a method to improve the stability and pharmacokinetics of antibody-beta G conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect beta G activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity The, enzyme was found to be stable in serum regardless of PEG modification. PEG-modified beta G was coupled via a thioether bond to mAb RH1, an IgG(2a) antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-beta G-3PEG), 5.2 (RH1-beta G-5PEG) or 9.8 (RH1-beta G-10PEG) PEG molecules per beta G with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-beta G. In contrast to the rapid serum clearance of RH1-beta G observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-beta G-3PEG and RH1-beta G-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-beta G. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-beta G-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-beta G conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.