溶瘤病毒的免疫学机制及临床研究进展

溶瘤病毒（oncolytic virus,OVs）历经百年发展,应用于当前最具潜力的肿瘤免疫疗法。它主要是天然的或基因修饰的DNA病毒和RNA病毒。近年来随着基因工程技术的飞跃发展,经基因改造的溶瘤病毒在肿瘤治疗领域取得很大进展,很多不同类型的病毒（包括单纯疱疹病毒、腺病毒、痘病毒、麻疹病毒和呼肠孤病毒等）正处于临床前研究、临床试验阶段或已批准上市,显示了良好的安全性和临床疗效。普遍认为溶瘤病毒靶向杀伤肿瘤细胞是通过选择性在肿瘤细胞内自我复制,最终裂解肿瘤细胞,同时可激发机体的免疫应答反应,进而增强抗肿瘤免疫效果,靶向杀伤肿瘤细胞而对正常细胞无明显影响。运用基因重组技术将溶瘤病毒与免疫检查点相结合以及肿瘤免疫联合疗法的兴起和不断进步,使溶瘤病毒的应用更加广泛,但仍存在病毒靶向性、安全性、给药途径等瓶颈问题。本文综述了溶瘤病毒的发展史、病毒分类、不同类型溶瘤病毒产品的临床研究进展、溶瘤病毒靶向杀伤肿瘤的免疫学机制及未来发展面临的挑战与展望等。     

溶瘤病毒靶向治疗肿瘤的策略

近年来,随着国内外几款溶瘤病毒制剂的相继上市,溶瘤病毒疗法成为肿瘤免疫治疗的焦点。溶瘤病毒可选择性感染并裂解肿瘤细胞,同时释放肿瘤相关抗原激活机体的抗肿瘤免疫反应,达到杀伤肿瘤细胞和抑制肿瘤生长的目的。溶瘤病毒对肿瘤的靶向杀伤作用决定了其安全性和溶瘤效果。为了开发出安全高效的溶瘤病毒,目前主要采用以下策略：利用某些病毒载体对肿瘤细胞的天然靶向性,使溶瘤病毒选择性地在肿瘤细胞内复制并杀伤肿瘤细胞;或者对病毒基因组进行缺失和插入等修饰,通过靶向肿瘤细胞特异性表面受体、胞内信号通路或者肿瘤微环境等提高溶瘤病毒的肿瘤靶向性。其中,肿瘤微环境中的低氧状态、新血管生成以及免疫抑制状态等都可成为溶瘤病毒的靶点。而溶瘤病毒通过表达细胞因子和免疫检查点抑制剂,或者与CAR-T细胞联合作用,靶向调节肿瘤微环境中免疫抑制状态,成为提高溶瘤病毒肿瘤靶向性的常用方法。本文将对以上溶瘤病毒靶向治疗肿瘤策略的研究进展进行综述。     

Developing oncolytic viruses for clinical use: A consortium approach

The use of oncolytic viruses forms an appealing approach for cancer treatment. On the one hand the viruses replicate in, and kill, tumor cells, leading to their intra-tumoral amplification. On the other hand the viral infection will activate virus-directed immune responses, and may trigger immune responses directed against tumor cells and tumor antigens. To date, a wide variety of oncolytic viruses is being developed for use in cancer treatment. While the development of oncolytic viruses has often been initiated by researchers in academia and other public institutions, a large majority of the final product development and the testing of these products in clinical trials is industry led. As a consequence relatively few pre-clinical and clinical studies evaluated different oncolytic viruses in competitive side-by-side preclinical or clinical studies. In this review we will summarize the steps and considerations essential in the development and characterization of oncolytic viruses, and describe our multidisciplinary academic consortium, which involves a dozen departments in three different Dutch universities, collaborating in the development of oncolytic viruses. This consortium has the ambition to develop a small series of oncolytic viruses and to evaluate these in various cancers.     

Mechanisms of measles virus oncolytic immunotherapy

The study of measles virus (MeV) as a cancer immunotherapeutic was prompted by clinical observations of leukemia and lymphoma regressions in patients following measles virus infection in the 1970s and 1980s. Since then, numerous preclinical studies have confirmed the oncolytic activity of MeV vaccine strains as well as their potential to promote long-lasting tumor-specific immune responses. Early clinical data indicate that some of these effects may translate to the treatment of cancer patients. In this review, we provide a structured summary of current evidence for the anti-tumor immune activity of oncolytic MeV. We start with an overview of MeV oncolysis and MeV-induced immunogenic cell death. Next, we relate findings on MeV-mediated activation of antigen-presenting cells, T cell priming and effector mechanisms to the cancer immunity cycle. We discuss additional factors in the tumor microenvironment which are modulated by MeV treatment as well as the role of anti-viral immunity. Based on these findings, we highlight avenues for rational enhancement of oncolytic MeV immunotherapy by vector engineering. We further point to advantages and drawbacks of experimental models and propose areas warranting promising research. Lastly, we review the available immunomonitoring data from several Phase I clinical trials. While this review presents data for MeV, the concepts and principles introduced herein apply to other oncolytic viruses, providing a framework to assess novel cancer immunotherapies.     

溶瘤麻疹病毒的临床研究与转化进展

肿瘤已成为危及全人类生命的重大疾病,虽然常规治疗手段如手术及放疗/化疗等不断发展,但对某些复发、难治性恶性肿瘤仍然束手无策,亟需安全、有效可行的治疗手段。在肿瘤基因治疗领域,能在肿瘤细胞内大量自我复制并选择性杀伤肿瘤细胞的溶瘤病毒(Oncolytic virus)逐渐崭露头角,目前溶瘤病毒抗瘤治疗备受关注。其中溶瘤麻疹病毒疫苗株(MV-Edm)因其可靠的安全性和优良的溶瘤效果已进入几项临床试验,为推动其临床转化奠定了基础,期望为肿瘤患者带来治疗上的突破。本文就目前溶瘤麻疹病毒的临床研究与转化进展的相关研究成果进行综述。     

United virus: The oncolytic tag-team against cancer!

There is an urgent need for innovative therapeutic strategies to treat aggressive metastatic cancers that are incurable with standard therapeutic approaches. Novel treatment strategies like oncolytic virotherapy have led, in some cases, to impressive effects on disease progression in human trials, suggesting that approval of an oncolytic virus therapeutic is on the horizon. While combinations of oncolytic viruses with small molecules are already being tested and have shown promise, we propose that even greater therapeutic synergies could be achieved through rational design of complementary virus therapeutics. In this review, we discuss rational chemical and biological combination strategies to enhance oncolytic virotherapy highlighting the promising combination of vaccinia and vesicular stomatitis oncolytic viruses.     

Oncolytic adenoviruses in anticancer therapy: Current status and prospects

Lytic virus infection results in production of a virus progeny and lysis of the infected cell. Tumor cells are usually more sensitive to virus infection. Studies indicate that viral oncolysis provides a promising alternative approach to cancer therapy. The ability of viruses to selectively kill cancer cells is long known, but construction of virus variants with an improved therapeutic potential was impossible until recent advances in virus and cell molecular biology and the development of modern methods for directed modification of viruses. Adenoviruses are one of the best studied models of oncolytic viruses. These DNA viruses are convenient for genetic manipulation and show minimal pathogenicity. The review summarizes the data on the directions and approaches to generation of highly efficient variants of oncolytic adenoviruses. The approaches include introduction of directed genetic modifications into the virus genome, accelerated selection of oncolytic virus variants following treatment with mutagens, the use of adenoviruses as vectors to introduce therapeutic gene products, optimization of viral delivery systems, minimization of the negative effects from the host immune system, etc. The dynamic development of studies in the field holds promise that many variants of oncolytic adenoviruses will find clinical application in the nearest future.     

Systemic therapy for cervical cancer with potentially regulatable oncolytic adenoviruses

Clinical trials have confirmed the safety of selectively oncolytic adenoviruses for treatment of advanced cancers. However, increasingly effective viruses could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. We analyzed viruses containing the cyclooxygenase-2 (Cox-2) or vascular endothelial growth factor (VEGF) promoter for controlling replication. Anti-inflammatory agents can lower Cox-2 protein levels and therefore we hypothesized that also the promoter might be affected. As Cox-2 modulates expression of VEGF, also the VEGF promoter might be controllable. First, we evaluated the effect of anti-inflammatory agents on promoter activity or adenovirus infectivity in vitro. Further, we analyzed the oncolytic potency of the viruses in vitro and in vivo with and without the reagents. Moreover, the effect of on virus replication was analyzed. We found that RGD-4C or Ad5/3 modified fibers improved the oncolytic potency of the viruses in vitro and in vivo. We found that both promoters could be downregulated with dexamethasone, sodium salicylate, or salicylic acid. Oncolytic efficacy correlated with the promoter activity and in vitro virus production could be abrogated with the substances. In vivo, we saw good therapeutic efficacy of the viruses in a model of intravenous therapy of metastatic cervical cancer, but the inhibitory effect of dexamethasone was not strong enough to provide significant differences in a complex in vivo environment. Our results suggest that anti-inflammatory drugs may affect the replication of adenovirus, which might be relevant in case of replication associated side effects.     

可复制性单纯疱疹病毒在抗肿瘤方面的应用

单纯疱疹病毒是肿瘤生物治疗中常用的病毒载体之一,可复制性单纯疱疹病毒以其溶瘤效率高、特异性好、可行性强成为近年来研究的热点。其中对溶瘤性单纯疱疹病毒突变株G207的研究开展得早,其溶瘤效果、靶向性及安全性都得到了确认,这也带动了可复制性单疱病毒应用的发展,目前已研究出多种溶瘤单纯疱疹病毒突变株。本文就近几年可复制性单纯疱疹病毒在抗肿瘤方面的研究现状加以综述,以探讨其临床治疗肿瘤的潜在价值及可行性。     

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading

Oncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.     

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.     

Oncolytic viruses as experimental treatments for malignant gliomas: Using a scourge to treat a devil

The concept of oncolytic viral therapy has a century-old history, but only within the last 20 years have oncolytic viruses been considered for the treatment of brain cancers. Viruses such as herpes, measles, and vaccinia have all been known to cause devastating cases of neurological disease in humans, yet these ‘scourges’ are now being harnessed in such a way that they prove very useful as cancer therapeutics. There have been 8 formal clinical trials and 3 case studies using oncolytic viruses to treat malignant glioma patients. Although some success has been reached with oncolytic therapy, overall it has fallen short of expectations. In this review we analyze the results of these trials and bring to light some of the limitations and pitfalls of this therapy, as well as present some promising preclinical work that has been proposed to circumvent such problems.     

靶向宿主代谢重编程的溶瘤病毒调控策略

溶瘤病毒是一类天然的或经过基因编辑后能特异性在肿瘤细胞中复制、发挥抗肿瘤效应的病毒。溶瘤病毒的抗肿瘤效应主要通过以下两个方面实现：a. 直接的溶瘤效应，例如诱导肿瘤细胞发生凋亡、促使细胞裂解等；b. 溶瘤病毒作为一种激活免疫的药物，通过诱导机体产生强烈的抗肿瘤免疫，达到清除肿瘤的目的。溶瘤病毒疗法作为免疫疗法的一个重要分支，因其具有肿瘤特异性，便于基因改造等优点，成为该领域的研究热点。截至目前，处在临床实验招募和完成阶段的溶瘤病毒疗法虽然已达100多例，但已批准上市的产品仅有4款。溶瘤疗法应用于肿瘤治疗领域还面临着诸多挑战。因此，系统性回顾溶瘤病毒的改造策略，深入了解溶瘤病毒的生物学过程显得尤为必要。病毒依赖于宿主完成复制、增殖过程，其生物学过程与宿主的代谢状态密切相关。肿瘤的标志性特征为代谢重编程，即肿瘤细胞重新构建代谢网络以满足指数生长和增殖的需求并防止氧化应激的过程。通常包括糖酵解的增强和谷氨酰胺分解，以及线粒体功能和氧化还原稳态的变化。通过靶向宿主代谢重编程增强溶瘤病毒的复制、溶瘤能力是当前极具前景的方向。本文综述溶瘤病毒的临床应用现状及与代谢相关的调控机制，为进一步开发新型溶瘤病毒以及联用方式提供新的思路。     

Clinical trials with oncolytic reovirus: Moving beyond phase I into combinations with standard therapeutics

It is time for those working on oncolytic viruses to take stock of the status of the field. We now have at our disposal an array of potential therapeutic agents, and are beginning to conduct early-phase clinical trials in patients with relapsed/metastatic cancers. By drawing on lessons learned during the development of other biological therapies, such as monoclonal antibodies and targeted small molecule inhibitors, we are now in a position to chart the course of the next wave of trials that will go beyond the phase I studies of safety and feasibility. In this article we review our approach to the development of oncolytic viruses as cancer therapeutics. In doing so, we emphasise the fact that this process is modular and involves multiple iterative steps between the laboratory and the clinic. Ultimately, at least in the medium term, the future of oncolytic virotherapy lies in combination regimens with standard anti-cancer agents such as radiation and chemotherapy.     

Oncolytic Newcastle disease virus reduces growth of cervical cancer cell by inducing apoptosis

Although Oncolytic viruses have been regarded as a promising tool for targeted therapy of cancer, accomplishing high efficacy and specificity with this strategy is challenging. Oncolytic virotherapy is one of the novel therapeutic methods recently used for the therapy of human malignancies. Cervical cancer is on the major public health problem and the second most common cause of cancer death among females in less developed countries. The aim of this study was mainly to determine the apoptosis effect of oncolytic Newcastle disease virus (NDV) in TC-1 cell line.In the current study, the oncolytic NDV, vaccine strain LaSota, was used to infect murine TC-1 cells of human papillomavirus (HPV)-associated carcinoma which expressing human papillomavirus 16 (HPV-16) E6/E7 antigens in vitro. The effectiveness of NDV for cervical cancer cell line was investigated by evaluating the antitumor activity of oncolytic NDV and the involved mechanisms. Antitumor activities of oncolytic NDV were assessed by cell proliferation (MTT) and lactate dehydrogenase (LDH) release analysis. In addition, molecular changes of early stage of apoptosis and the role of reactive oxygen species (ROS) were analyzed by flow cytometry and Western Blot in NDV-treated TC-1 cells.The results showed that NDV treatment significantly decreased the viability of a TC-1 cell line and suppressed the growth by inducing apoptotic cell death. In addition, we demonstrated that NDV-induced apoptosis of TC-1 cells is mediated by ROS production. In summary, our findings suggest that oncolytic NDV is a possible therapeutic candidate as a selective antitumor agent for the treatment of cervical cancer.     

Measles virus: a future therapeutic agent in oncology?

Measles is a potential lethal disease, justifying large immunization campaigns. Attenuated strains are used in immunization with very good safety records. Interestingly, following clinical observations of tumor regressions after measles infection, preclinical and clinical studies have highlighted the therapeutic potential of attenuated strains of measles. The aim of this review is to explain how these viruses can selectively infect and kill cancer cells, and how this selectivity can be improved. We will detail the therapeutic strategies under development, in particular the combination of viruses with chemotherapy and radiation therapy. Furthermore, the engineering of measles viruses encoding the sodium/iodide symporter could enable virus-directed radio-isotope therapy. Antiviral immunity could be a limit of measles therapy. We will highlight the promising combinations with immunosuppressive drugs and innovative strategies using infected cell carriers, aiming at circumventing the immune response and paving the way to future clinical trials.     

Combining oncolytic virotherapy and tumour vaccination

The interactions between the immune system, a malignant tumour and an oncolytic virus are complex and poorly understood. For oncolytic viruses to become successful therapeutics we need to better understand these interactions and identify strategies to take advantage of defects in the innate immune response within tumours and avoid cellular anti-viral responses while capitalizing on anti-tumoural immunity. In this review we will discuss the evidence for the induction of tumour-specific immune responses by oncolytic viruses as well as by cancer vaccines. We will then describe some of the barriers to successful cancer immunotherapy, and finally we will outline a strategy for enhancing anti-tumoural immunity while reducing anti-viral immunity by combining tumour vaccination with oncolytic viral therapy.     

The combination therapy of oncolytic HSV-1 armed with anti-PD-1 antibody and IL-12 enhances anti-tumor efficacy

Cancer immunotherapy is a new therapeutic strategy for cancer treatment that targets tumors by improving or restoring immune system function. Therapies targeting immune checkpoint molecules have exerted potent anti-tumor effects and prolonged the overall survival rate of patients. However, only a small number of patients benefit from the treatment. Oncolytic viruses exert anti-tumor effects by regulating the tumor microenvironment and affecting multiple steps of tumor immune circulation. In this study, we engineered two oncolytic viruses that express mouse anti-PD-1 antibody (VT1093M) or mouse IL-12 (VT1092M). We found that both oncolytic viruses showed significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Importantly, the intratumoral combined injection with VT1092M and VT1093M inhibited growth of the primary tumor, prevented growth of the contralateral untreated tumor, produced a vaccine-like response, activated antigen-specific T cell responses and prolonged the overall survival rate of mice. These results indicate that combination therapy with the engineered oncolytic virus may represent a potent immunotherapy strategy for cancer patients, especially those resistant to PD-1/PD-L1 blockade therapy.     

Kinase inhibitors with viral oncolysis: Unmasking pharmacoviral approaches for cancer therapy

There are more than 500 kinases in the human genome, many of which are oncogenic once constitutively activated. Fortunately, numerous hyperactive kinases are druggable, and several targeted small molecule kinase inhibitors have demonstrated impressive clinical benefits in cancer treatment. However, their often cytostatic rather than cytotoxic effect on cancer cells, and the development of resistance mechanisms, remain significant limitations to these targeted therapies. Oncolytic viruses are an emerging class of immunotherapeutic agents with a specific oncotropic nature and excellent safety profile, highlighting them as a promising alternative to conventional therapeutic modalities. Nonetheless, the clinical efficacy of oncolytic virotherapy is challenged by immunological and physical barriers that limit viral delivery, replication, and spread within tumours. Several of these barriers are often associated with oncogenic kinase activity and, in some cases, worsened by the action of oncolytic viruses on kinase signaling during infection. What if inhibiting these kinases could potentiate the cancer-lytic and anti-tumour immune stimulating properties of oncolytic virotherapies? This could represent a paradigm shift in the use of specific kinase inhibitors in the clinic and provide a novel therapeutic approach to the treatment of cancers. A phase III clinical trial combining the oncolytic Vaccinia virus Pexa-Vec with the kinase inhibitor Sorafenib was initiated. While this trial failed to show any benefits over Sorafenib monotherapy in patients with advanced liver cancer, several pre-clinical studies demonstrate that targeting kinases combined with oncolytic viruses have synergistic effects highlighting this strategy as a unique avenue to cancer therapy. Herein, we review the combinations of oncolytic viruses with kinase inhibitors reported in the literature and discuss the clinical opportunities that represent these pharmacoviral approaches.     

Cancer and viruses: a double-edged sword

Oncovirus, synonymously called a 'tumour virus', is a virus that can cause cancer. An oncolytic virus preferentially infects the host's cancer cells and lyses them, causing tumour destruction, and is thus referred to as a 'cancer killing virus'. With an estimated 11% of cancer-associated deaths caused by oncoviruses and the possibility that many cancers may be treated by using oncolytic viruses, the role of viruses in cancer may be viewed as a double-edged sword. A total of seven human cancer viruses have been identified as oncoviruses, having been associated with various cancers. Conversely, a large number of oncolytic viruses have shown great potential towards the treatment of certain types of cancer. Proteomics has now been applied towards understanding the complex interplay that exists between oncoviruses and the immune responses that serve to prevent oncoviral diseases. This review attempts to summarise the neoplastic potential of human tumour associated viruses and associated vaccine successes. The potential use of oncolytic viruses for the therapeutic intervention of cancer will also be discussed. Finally, this review will discuss the enormous potential of proteomics technology in the field of oncovirology.