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.

血液检测是转化研究中监测溶瘤病毒疗法（oncolytic virotherapy）及其他生物疗法的必要、易操作且低成本的替代方案。本研究评估了三种有望用作生物体液（biological fluids）生物标志物的候选蛋白：两种分别来自大肠杆菌（E. coli）的葡萄糖醛酸苷酶（glucuronidases）GusA与葡萄球菌属（Staphylococcus sp.）RLH1菌株的GusPlus，以及来自克氏纺锤镖水母（Gaussia princeps）的荧光素酶（luciferase）GLuc。研究人员将编码这三种蛋白的三个基因分别置于相同启动子（promoter）的调控下，插入痘病毒（vaccinia virus）GLV-1h68的基因组中。随后利用获得的三种重组病毒（recombinant viruses）分别感染体外培养的肿瘤细胞与裸鼠（nude mice）体内的人源肿瘤异种移植瘤（human tumor xenografts）。与主动分泌的GLuc不同，胞质葡萄糖醛酸苷酶GusA和GusPlus仅在病毒介导的溶瘤作用（oncolysis）发生时，才会被释放至细胞上清液（supernatants）中。在受控的实验条件下，GusPlus对酶活性的检测灵敏度最高，当样本中GusPlus含量仅为1 pg/ml时即可检出；其次为GusA（25 pg/ml），最后为GLuc（≥375 pg/ml）。出乎意料的是，尽管GusA的比活性（specific activity）低于GusPlus，但注射了编码GusA的病毒菌株的荷瘤小鼠血清中的底物转化率，显著高于注射GusPlus编码病毒的小鼠。该现象归因于GusA在血流中的半衰期（half-life）分别比GusPlus和GLuc长3.2倍和16.2倍，因此相较于另外两种酶，GusA是更为灵敏的病毒复制监测标志物。由于标记基因的酶活性与病毒滴度（virus titer）之间存在良好的相关性，研究人员得出结论：体液中的生物标志物蛋白含量可半定量地反映感染肿瘤内的病毒载量，该结论通过低光成像（low light imaging）实验得到了验证。在本次测试的三种标志物中，GusA被证实为监测溶瘤病毒疗法最为可靠的生物标志物。