Lin Amy H, Burrascano Cindy, Pettersson Par L, Ibañez Carlos E, Gruber Harry E, Jolly Douglas J
Tocagen Inc., San Diego, California, USA.
Tocagen Inc., San Diego, California, USA
J Virol. 2014 Sep 1;88(17):10066-77. doi: 10.1128/JVI.02300-13. Epub 2014 Jun 25.
We developed a Moloney mouse leukemia virus (MLV)-based retroviral replicating vector (RRV), Toca 511, which has displayed tumor specificity in resected brain tumor material and blood in clinical trials. Here, we investigated the interaction between Toca 511 and human host cells, and we show that RRVs do not induce type I interferon (IFN) responses in cultured human tumor cells or cultured human primary cells. However, exogenous type I IFN inhibited RRV replication in tumor cells and induced IFN-regulated genes, albeit at a lower level than in primary cells. Unexpectedly, RRVs did not induce IFN-α production upon incubation in vitro with human plasmacytoid dendritic cells (pDCs), whereas lentivirus vector and heat-treated RRVs did. Coincubation of RRVs with heat-treated RRVs or with lentivirus vector suppressed IFN-α production in pDCs, suggesting that native RRV has a dominant inhibitory effect on type I IFN induction. This effect is sensitive to trypsin treatment. In addition, heat treatment inactivated that activity but exposed an immune-stimulatory activity. The immune-stimulating component is sensitive to deglycosidases, trypsin, and phospholipase C treatment. Experiments with retroviral nonreplicating vectors and virus-like particles demonstrated that the immunosuppressive activity is not associated with the amphotropic envelope or the glyco-Gag protein. In summary, our data provide evidence that RRVs do not directly trigger type I IFN responses in IFN-responsive tumor cells. Moreover, RRVs appear to carry a heat-labile component that actively suppresses activation of cellular innate immune responses in pDCs. Inhibition of IFN induction by RRVs and the reduced response to IFN should facilitate tumor-specific infection in vivo.
RRVs have a convincing preference for replicating in tumor cells in animal models, and we observed similar preferences in the initial treatment of human glioblastoma patients. This study investigates the basis for the interaction between RRV and human host cells (tumor versus nontumor) in vitro. We found that RRVs do not trigger an IFN-α/β response in tumor cells, but the cells are capable of responding to type I IFNs and of producing them when stimulated with known agonists. Surprisingly, the data show that RRVs can actively inhibit induction of cellular innate immunity and that this inhibitory activity is heat labile and trypsin sensitive and not attributable to the envelope protein. These data partially explain the observed in vivo tumor specificity.
我们开发了一种基于莫洛尼鼠白血病病毒（MLV）的逆转录病毒复制载体（RRV），即Toca 511，它在临床试验中已在切除的脑肿瘤组织和血液中显示出肿瘤特异性。在此，我们研究了Toca 511与人类宿主细胞之间的相互作用，结果表明RRV在培养的人类肿瘤细胞或培养的人类原代细胞中不会诱导I型干扰素（IFN）反应。然而，外源性I型干扰素抑制了肿瘤细胞中的RRV复制并诱导了IFN调节基因，尽管其水平低于原代细胞。出乎意料的是，RRV在体外与人浆细胞样树突状细胞（pDC）孵育时不会诱导IFN-α产生，而慢病毒载体和热处理的RRV则会。RRV与热处理的RRV或慢病毒载体共同孵育可抑制pDC中的IFN-α产生，这表明天然RRV对I型干扰素诱导具有显性抑制作用。这种作用对胰蛋白酶处理敏感。此外热处理使该活性失活，但暴露了一种免疫刺激活性。免疫刺激成分对糖苷酶、胰蛋白酶和磷脂酶C处理敏感。用逆转录病毒非复制载体和病毒样颗粒进行的实验表明，免疫抑制活性与嗜异性包膜或糖基化Gag蛋白无关。总之，我们的数据表明RRV在IFN反应性肿瘤细胞中不会直接触发I型干扰素反应。此外，RRV似乎携带一种热不稳定成分，可积极抑制pDC中细胞固有免疫反应激活。RRV对干扰素诱导的抑制以及对干扰素反应的降低应有助于体内肿瘤特异性感染。
RRV在动物模型中对肿瘤细胞的复制具有令人信服的偏好，并且我们在人类胶质母细胞瘤患者的初始治疗中也观察到了类似的偏好。本研究在体外研究了RRV与人类宿主细胞（肿瘤细胞与非肿瘤细胞）之间相互作用的基础。我们发现RRV在肿瘤细胞中不会触发IFN-α/β反应，但这些细胞能够对I型干扰素作出反应，并在受到已知激动剂刺激时产生干扰素。令人惊讶的是，数据表明RRV可以积极抑制细胞固有免疫的诱导，并且这种抑制活性是热不稳定的且对胰蛋白酶敏感，并且不归因于包膜蛋白。这些数据部分解释了观察到的体内肿瘤特异性。
