Methoxypoly(
ethylene glycol) (PEG) modification of Escherichia coli
beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of
glucuronide prodrugs at
tumor cells. Introduction of 3 PEG molecules did not affect betaG 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 betaG was coupled via a
thioether bond to mAb RH1, an
IgG2a antibody that binds to the surface of AS-30D
hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined
antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater
tumor accumulation than RH1-betaG.
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-betaG-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-betaG conjugate and
BHAMG delayed
tumor growth and cured two of six mice while treatment with pHAM or
BHAMG alone was ineffective.