Z. Sheng Guo

Oncolytic virotherapy : Molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will highlight some avenues deserving further study in order to achieve the ultimate goals of utilizing various OVs for effective cancer treatment.

The Enhanced Tumor Selectivity of an Oncolytic Vaccinia Lacking the Host Range and Antiapoptosis Genes SPI-1 and SPI-2

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

Chemokine Expression From Oncolytic Vaccinia Virus Enhances Vaccine Therapies of Cancer

Tumor vaccines can induce robust immune responses targeting tumor antigens in the clinic, but antitumor effects have been disappointing. One reason for this is ineffective tumor infiltration of the cytotoxic T lymphocytes (CTLs) produced. Oncolytic viruses are capable of selectively replicating within tumor tissue and can induce a strong immune response. We therefore sought to determine whether these therapies could be rationally combined such that modulation of the tumor microenvironment by the viral therapy could help direct beneficial CTLs induced by the vaccine. As such, we examined the effects of expressing chemokines from oncolytic vaccinia virus, including CCL5 (RANTES), whose receptors are expressed on CTLs induced by different vaccines, including type-1-polarized dendritic cells (DC1). vvCCL5, an oncolytic vaccinia virus expressing CCL5, induced chemotaxis of lymphocyte populations in vitro and in vivo, and displayed improved safety in vivo. Interestingly, enhanced therapeutic benefits with vvCCL5 in vivo correlated with increased persistence of the viral agent exclusively within the tumor. When tumor-bearing mice were both vaccinated with DC1 and treated with vvCCL5 a further significant enhancement in tumor response was achieved which correlated with increased levels of tumor infiltrating lymphocytes. This approach therefore represents a novel means of combining biological therapies for cancer treatment.

Oncolytic Virus and Anti–4-1BB Combination Therapy Elicits Strong Antitumor Immunity against Established Cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.

High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-α, IL-6, TNF-α, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-γ-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.

T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy

Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. This lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. We reasoned that redirecting T cells to the tumor has the potential to improve the antitumor activity of oncolytic VVs. We, therefore, constructed a VV encoding a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 (EphA2-T-cell engager-armed VV (EphA2-TEA-VV)). In vitro, EphA2-TEA-VVs ability to replicate and induce oncolysis was similar to that of unmodified virus. However, only tumor cells infected with EphA2-TEA-VV induced T-cell activation as judged by the secretion of interferon-γ and interleukin-2. In coculture assays, EphA2-TEA-VV not only killed infected tumor cells, but in the presence of T cells, it also induced bystander killing of noninfected tumor cells. In vivo, EphA2-TEA-VV plus T cells had potent antitumor activity in comparison with control VV plus T cells in a lung cancer xenograft model. Thus, arming oncolytic VVs with T-cell engagers may represent a promising approach to improve oncolytic virus therapy.

Gene transfer: the challenge of regulated gene expression

Gene therapy is expected to have a major impact on human healthcare in the future. However, precise regulation of therapeutic gene expression in vivo is still a challenge. Natural and synthetic enhancer-promoters (EPs) can be utilized to drive gene transcription in a temporal, spatial or environmental signal-inducible manner in response to heat shock, hypoxia, radiation, chemotherapy, epigenetic agents or viral infection. To allow tightly regulated expression, a regulatable gene-expression system can also be implemented. Most of these systems are based on small molecule (drug)-responsive artificial transactivators. In this review, we aim to provide a brief overview of the classes of EPs and regulatable systems, along with lessons learned from these studies. We highlight the potential applications in gene transfer, gene therapy for cancer and genetic disease and the future challenges for clinical applications.

Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

Both anti-PD1/PD-L1 therapy and oncolytic virotherapy have demonstrated promise, yet have exhibited efficacy in only a small fraction of cancer patients. Here we hypothesized that an oncolytic poxvirus would attract T cells into the tumour, and induce PD-L1 expression in cancer and immune cells, leading to more susceptible targets for anti-PD-L1 immunotherapy. Our results demonstrate in colon and ovarian cancer models that an oncolytic vaccinia virus attracts effector T cells and induces PD-L1 expression on both cancer and immune cells in the tumour. The dual therapy reduces PD-L1+ cells and facilitates non-redundant tumour infiltration of effector CD8+, CD4+ T cells, with increased IFN-γ, ICOS, granzyme B and perforin expression. Furthermore, the treatment reduces the virus-induced PD-L1+ DC, MDSC, TAM and Treg, as well as co-inhibitory molecules-double-positive, severely exhausted PD-1+CD8+ T cells, leading to reduced tumour burden and improved survival. This combinatorial therapy may be applicable to a much wider population of cancer patients.

Three epigenetic drugs up-regulate homeobox gene Rhox5 in cancer cells through overlapping and distinct molecular mechanisms.

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2′-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

Local Administration of TLR Ligands Rescues the Function of Tumor-Infiltrating CD8 T Cells and Enhances the Antitumor Effect of Lentivector Immunization

Cancer vaccines, to date, have shown limited effect to control the growth of established tumors due largely to effector failure of the antitumor immune responses. Tumor lesion is characterized as chronic indolent inflammation in which the effector function of tumor-infiltrating lymphocytes (TILs) is severely impaired. In this study, we investigated whether the effector function of CD8 TILs could be rescued by converting the chronic inflammation milieu to acute inflammation within tumors. We found that injection of TLR3/9 ligands (polyI:C/CpG) into a tumor during the effector phase of lentivector (lv) immunization effectively rescued the function of lv-activated CD8 TILs and decreased the percentage of T regulatory within the tumor, resulting in a marked improvement in the antitumor efficacy of lv immunization. Mechanistically, rescue of the effector function of CD8 TILs by TLR3/9 ligands is most likely dependent on production, within a tumor, of type-1 IFN that can mature and activate tumor-infiltrating dendritic cells. The effector function of CD8 TILs could not be rescued in mice lacking intact type I IFN signaling. These findings have important implications for tumor immunotherapy, suggesting that type I IFN-mediated activation of tumor-infiltrating dendritic cells within a tumor will most likely restore/enhance the effector function of CD8 TILs and thus improve the antitumor efficacy of current cancer vaccines.