Drug-Encoded Biomarkers for Monitoring Biological Therapies

PLoS One. 2015 Sep 8;10(9):e0137573. doi: 10.1371/journal.pone.0137573. eCollection 2015.

Abstract

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers, Tumor / genetics*
  • Cell Line, Tumor
  • Escherichia coli / enzymology
  • Glucuronidase / biosynthesis
  • Glucuronidase / genetics*
  • Humans
  • Luciferases / biosynthesis
  • Luciferases / genetics*
  • Mice
  • Neoplasms / genetics
  • Neoplasms / therapy*
  • Neoplasms / virology
  • Oncolytic Virotherapy*
  • Oncolytic Viruses / genetics
  • Staphylococcus / enzymology
  • Vaccinia virus / genetics
  • Xenograft Model Antitumor Assays

Substances

  • Biomarkers, Tumor
  • Luciferases
  • Glucuronidase

Grant support

The work was supported by Genelux Corporation and a Service Grant awarded to the University of Würzburg, graduate stipends from the University of Würzburg. The publication costs were co-funded by the German Research Foundation (DFG) and the University of Würzburg in the funding program Open Access Publishing. The funder provided support in the form of salaries for authors (JS, QZ, AF, JC, UF, AAS), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.