Drug-Encoded Biomarkers for Monitoring Biological Therapies

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.

Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis

“…The quantitative β-glucuronidase assay using this GusA enzyme is a very promising and well established in our laboratory reporter system 27, 28, 29. It is very accurate, requires very small sample volume, uses inexpensive substrates, and could be carried out in a very short time.…”

“…The quantitative β-glucuronidase assay using this GusA enzyme is a very promising and well established in our laboratory reporter system. 27 , 28 , 29 It is very accurate, requires very small sample volume, uses inexpensive substrates, and could be carried out in a very short time. Importantly, excellent correlations between levels of CTLA4 scAb, GusA, and viral titers, assayed separately by ELISA, β-glucuronidase assay, and standard plaque assay, respectively, in A549 cultures and humanized-mouse-derived human A549 tumors infected with GLV-1h376 were observed.…”

“…We further applied this mouse model to characterize the human immune cells present in tumors and organs of virus-treated or control mice, as well as to analyze the effect of a rVACV-encoded human CTLA4-blocking single-chain antibody (CTLA4 scAb) on the activation status of the human T cells. Further, we studied the potential of β-glucoronidase assay, well-established in our laboratory, 27 , 28 , 29 to be used as a less time- and labor-consuming alternative for determination of viral titer and CTLA4 scAb concentration in humanized-mouse-derived samples instead of replication assay and ELISA.…”

Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

“…Reporter-gene assays have helped the pox virologists in basic research, for example for the study of the location, structure and function of many VACV proteins during the infection cycle and their interaction with proteins of the host cell [ 44 , 70 ]. As shown in Dvoracek and Shors [ 63 ], the GUS reporter gene was used for deleting the D9R viral protein and selecting the recombinant viruses, with the aim to understand the role of this protein in the viral life cycle.…”

“…There are several in vivo applications for recombinant reporter-expressing viruses. For example, in virulence studies, the use of labeled viruses allows us to follow the viral pathogenicity and detect in which organs the viral replication and dissemination occur [ 70 , 76 ]. For example, Zaitseva et al [ 69 ] used the recombinant VACV Western Reverse strain (WR)-LUC to analyze the viral spread in vivo for several days reducing the number of mice used.…”