Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

Eur J Pharm Sci. 2019 Jan 15:127:79-91. doi: 10.1016/j.ejps.2018.10.014. Epub 2018 Oct 19.


Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, 'biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.

Keywords: ADEPT; Antibody directed enzyme prodrug therapy; CPG2; Cancer; Carboxypeptidase G2; HSA; HSA-glucarpidase; Human serum albumin; PEGylated glucarpidase; PEGylation.

MeSH terms

  • Antibodies / administration & dosage*
  • Antibodies / chemistry
  • Antimetabolites, Antineoplastic / pharmacology*
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Enzyme Therapy
  • Humans
  • Hydrolysis
  • Leukocytes, Mononuclear / drug effects
  • Methotrexate / pharmacology*
  • Neoplasms / drug therapy
  • Polyethylene Glycols / administration & dosage*
  • Polyethylene Glycols / chemistry
  • Prodrugs / administration & dosage*
  • Prodrugs / chemistry
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / chemistry
  • Serum Albumin, Human / administration & dosage*
  • Serum Albumin, Human / chemistry
  • Serum Albumin, Human / genetics
  • T-Lymphocytes / drug effects
  • gamma-Glutamyl Hydrolase / administration & dosage*
  • gamma-Glutamyl Hydrolase / chemistry


  • Antibodies
  • Antimetabolites, Antineoplastic
  • Prodrugs
  • Recombinant Proteins
  • glucarpidase
  • Polyethylene Glycols
  • gamma-Glutamyl Hydrolase
  • Methotrexate
  • Serum Albumin, Human