Drug-encoded Biomarkers for Monitoring Biological Therapies

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

Standard assays of purified marker enzymes.

<p>Activity vs enzyme concentration of glucuronidase assays (GusPlus upper chart; GusA middle) with the fluorogenic substrates Cl-MUGlcU, MUGlcU, (CUGlcU was also tested and the results were comparable to MUGlcU, data not shown), and FDGlcU, as well as with the luminogenic GusJuice in human serum are compared. The most sensitive assays for GusPlus and GusA with Cl-MUGlcU and GLuc with the Pierce Gaussia luciferase Glow Assay kit are compared (lower chart). All data points considered were above the limits of detection, as determined for each individual assay. Data points determined to be in the linear range are shown in black while all other data points are shown in grey.</p

Purification of His-tagged GusPlus.

<p><b>A)</b> GusPlus expression was induced in E. coli BL21 pET-28a-GusPlus and the bacteria were harvested by centrifugation (pellet) after overnight culture at room temperature. The bacteria were then lysed (lysate) and loaded on Ni-NTA beads onto which His-tagged GusPlus bound preferentially (GusPlus was clearly reduced in the flow through fraction). The Ni-NTA column was washed (wash fraction) and the beads in the column were shown to have high amounts of bound GusPlus (pre-elution beads). The elution fractions 2–5 contained highly enriched His-tagged GusPlus (post-elution beads). <b>B)</b> The combined, dialyzed elution fractions were analyzed by Coomassie-stained SDS-PAGE and determined to be 95.6% His-tagged GusPlus. Commercially available GusA was shown to be 49.3% pure by loading on a Coomassie-stained SDS-PAGE 523 ng of total protein of purified GusPlus (center lane) and 1014 ng of total protein of GusA (right lane). The equal intensities of the GusPlus and GusA bands demonstrated their differences in purity.</p

Glucuronidase activity as an indicator of cell death and rVACV titer.

<p><b>A)</b> The activities of lactate dehydrogenase and GusA (orange) or GusPlus (blue) in the supernatant of infected A549 (top panel), PC-3 (middle) and CV-1 (lower panel) cells correlated well (R²>0.8), which indicates that glucuronidase can be used as an indicator of (virus mediated) cell lysis. <b>B)</b> Due to its higher specific activity, GusPlus resulted in higher fluorescent product signal when compared to GusA specific signals in the same cells at the same time post infection. At the same time, the plaque forming units or glucuronidase ratios were similar (ratio about 1) in GLV-1h68 or GLV-1h408 infected cells. Data represent the average plus standard deviation of GLV-1h408/GLV-1h68-ratios obtained from simultaneously infected cells at different times post infection (n = 15). Asterisks indicate statistical differences (p<0.005) between fluorescence ratios and pfu- or glucuronidase ratios.</p

Glucuronidase assay substrates.

<p>Glucuronidase assay substrates.</p

Biomarker enzyme detection in tumor-bearing animals.

<p>A549 tumor-bearing animals were injected with GLV-1h68, GLV-1h351 or GLV-1h408. Every week, serum was isolated and tested for biomarker enzyme activity. <b>A)</b> GLV-1h351 injected mice were analyzed for the presence of GLuc and GusA activity. <b>B)</b> Significantly less GusPlus activity in serum of GLV-1h408 injected mice was observed at all time points compared to GusA in GLV-1h68 and GLV-1h351 injected mice (upper chart). At the same time points, no differences were observed in <i>Renilla</i> luciferase activity in GLV-1h68 and GLV-1h408 injected mice (lower chart). Grey crosses indicate that GLV-1h351 injected mice were not imaged for <i>Renilla</i> luciferase activity. <b>C)</b> Analysis of biomarker enzymes and viral titers in tumors at the end of the experiment. The activities of GLuc and GusPlus are clearly underrepresented relative to viral titers compared to GusA. <b>D)</b> Individual mouse data from samples that were obtained at the end of the experiment (28 dpi).</p

Glucuronidase assay in human blood and plasma.

<p>(A) GusA was spiked at varying concentrations into assay matrix (PBS with 2% FBS), human plasma and human blood respectively. A glucuronidase assay of the samples was performed using Cl-MUGlcU as substrate. The supernatant of spiked blood was used in the glucuroronidase assay after centrifugation (7 min, 3000 g). Mean plus standard deviation of 4 samples is shown with each concentration of glucuronidase tested in triplicate (n = 12 for each data point). (B) The data for 10⁴ pg/ml glucuronidase are shown in detail. The fluorescence signal derived from the blood sample is significantly higher compared to the signal that was obtained after spiking the same amount of GusA into identical volumes of assay matrix and plasma respectively. No significant difference was observed between the samples of the latter two groups.</p

Correlation between GLuc, GusA, and titer in GLV-1h351 and GLV-1h352 infected cells.

<p>A549 (triangles), CV-1 (circles) and PC-3 cells (squares) were infected with GLV-1h351 or GLV-1h352 using an MOI of 0.005. The supernatants and cell lysates were assayed for GLuc, GusA and plaque forming units at 8, 24, 48, 72, and 96 hours post infection. The “total” amount per well is the sum of the supernatant and cell lysate values. The grey line(s) in each graph indicates the linear range limit of each assay. Correlation coefficients (R²) were calculated using the data points above the linear range limits (indicated by crosses).</p

Properties of the different marker proteins and their assays.

<p>* Limit of detection for the described standard assays. For the glucuronidases, the detection limit was lower when incubation time was increased.</p><p>** The maximum concentration tested here was 1 μg/assay. According to Tannous, the linear range was at least 5 orders of magnitude [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137573#pone.0137573.ref018" target="_blank">18</a>].</p><p>Properties of the different marker proteins and their assays.</p