Efficient Clearance of Poly(ethylene glycol)-Modified Immunoenzyme with Anti-PEG Monoclonal Antibody for Prodrug Cancer Therapy

Abstract

The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.

Intravenous Administration of Polyethylene Glycol-Coated (PEGylated) Proteins and PEGylated Adenovirus Elicits an Anti-PEG Immunoglobulin M Response

“…22,32) A similar phenomenon was observed in PEGmodified poly lactide (PLA)-nanoparticles 33) and in PEG-modified micelles. 34) Roffler and co-workers 35,36) have generated anti-PEG IgM monoclonal antibody by immunization with an antibody-PEG-modified β-glucronidase (βG) and have shown that when intravenously injected the generated anti-PEG IgM induced an accelerated clearance of PEG-modified βG from blood circulation. Our current study demonstrated that the elicited anti-PEG IgM has a cross-reactivity between PEG grafted onto proteins and PEG on nanoparticular liposome (Fig.…”

“…Richter and Akerblom 27) proposed in an earlier study that the antigenic determinant of PEG may be a sequence of 6-7 -CH 2 CH 2 O-units. Roffler and co-workers 35,36) demonstrated that the monoclonal anti-PEG IgM generated by antibody-PEG-modified βG recognizes the repeating sequence (16 -CH 2 CH 2 O-units) of PEG. Thus, the anti-PEG IgM described in the present study may bind to the repeating sequence (-CH 2 CH 2 O-units) of PEG.…”

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

“…bG may allow both imaging and therapy of cancer with a single gene product as bG has demonstrated antitumor activity in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT). Immunoenzymes, formed by conjugating bG to antitumor antibodies, can selectively activate glucuronide prodrugs [29][30][31][32][33][34] allow accumulation of high drug concentrations at the tumor site, 35 produce bystander killing of antigen-negative tumor cells 31 and generate long-lasting protective immunity to subsequent tumor challenge. 34 Similarly, Brusselbach et al demonstrated that cell surface display of hbG for extracellular gene-directed enzyme prodrug therapy can produce strong bystander killing and potent antitumor activity.…”

PEGylation of therapeutic proteins

“…Presumably the methoxyl group in the PEG chain at the terminus remote from the linker to the protein [8, WO 2004/030617 A2] was identified as a source of antigenicity, which is rather surprising since methoxyl end-capped PEGs are generally used in modern marketed PEGylated biopharmaceuticals without reports on PEG immunogenicity. However, a few reports on induction of anti-PEG immune responses in the case of repeated administration of PEGylated liposomes [30,31] or PEGglucuronidase can be found in literature [32]. High levels of PEG used as an intravenous …”