Epitope-optimized alpha-fetoprotein genetic vaccines prevent carcinogen-induced murine autochthonous hepatocellular carcinoma.

UNLABELLED: Immunization with effective cancer vaccines can offer a much needed adjuvant therapy to fill the treatment gap after liver resection to prevent relapse of hepatocellular carcinoma (HCC). However, current HCC cancer vaccines are mostly based on native shared-self/tumor antigens that are only able to induce weak immune responses. In this study we investigated whether the HCC-associated self/tumor antigen of alpha-fetoprotein (AFP) could be engineered to create an effective vaccine to break immune tolerance and potently activate CD8 T cells to prevent clinically relevant carcinogen-induced autochthonous HCC in mice. We found that the approach of computer-guided methodical epitope-optimization created a highly immunogenic AFP and that immunization with lentivector expressing the epitope-optimized AFP, but not wild-type AFP, potently activated CD8 T cells. Critically, the activated CD8 T cells not only cross-recognized short synthetic wild-type AFP peptides, but also recognized and killed tumor cells expressing wild-type AFP protein. Immunization with lentivector expressing optimized AFP, but not native AFP, completely protected mice from tumor challenge and reduced the incidence of carcinogen-induced autochthonous HCC. In addition, prime-boost immunization with the optimized AFP significantly increased the frequency of AFP-specific memory CD8 T cells in the liver that were highly effective against emerging HCC tumor cells, further enhancing the tumor prevention of carcinogen-induced autochthonous HCC. CONCLUSIONS: Epitope-optimization is required to break immune tolerance and potently activate AFP-specific CD8 T cells, generating effective antitumor effect to prevent clinically relevant carcinogen-induced autochthonous HCC in mice. Our study provides a practical roadmap to develop effective human HCC vaccines that may result in an improved outcome compared to the current HCC vaccines based on wild-type AFP.

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.

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-VV's 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.

Lentivector prime and vaccinia virus vector boost generate high-quality CD8 memory T cells and prevent autochthonous mouse melanoma.

Most cancer vaccines, to date, fail to control established tumors. However, their application in preventing tumors is another question that is understudied. In the current study, we investigated the CD8 memory T cell responses of lentivector (lv) immunization and its potential to prevent melanoma using both transplantable B16 tumor and autochthonous melanoma models. We found that lv-expressing xenogenic human gp100 could induce potent CD8 responses that cross-react with mouse gp100. Importantly, the lv-primed CD8 response consisted of a high number of memory precursors and could be further increased by recombinant vaccinia virus vector (vv) boost, resulting in enhanced CD8 memory response. These long-lasting CD8 memory T cells played a critical role in immune surveillance and could rapidly respond and expand after sensing B16 tumor cells to prevent tumor establishment. Although CD8 response plays a dominant role after lv immunization, both CD4 and CD8 T cells are responsible for the immune prevention. In addition, we surprisingly found that CD4 help was not only critical for generating primary CD8 responses, but also important for secondary CD8 responses of vv boost. CD4 depletion prior to lv prime or prior to vv boost substantially reduced the magnitude of secondary CD8 effector and memory responses, and severely compromised the effect of cancer immune prevention. More importantly, the CD8 memory response from lv-vv prime-boost immunization could effectively prevent autochthonous melanoma in tumor-prone transgenic mice, providing a strong evidence that lv-vv prime-boost strategy is an effective approach for cancer immune prevention.

Chronic anti-inflammatory drug therapy inhibits gel-forming mucin production in a murine xenograft model of human pseudomyxoma peritonei.

BACKGROUND: Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. METHODS: The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. RESULTS: Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. CONCLUSIONS: Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

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.

Homeobox gene Rhox5 is regulated by epigenetic mechanisms in cancer and stem cells and promotes cancer growth.

BACKGROUND: Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene. FINDINGS: In Rhox5-silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo . CONCLUSIONS: Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.

Intratumoral expression of interleukin 23 variants using oncolytic vaccinia virus elicit potent antitumor effects on multiple tumor models via tumor microenvironment modulation.

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.

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.

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.

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.

De novo induction of a cancer/testis antigen by 5-aza-2'-deoxycytidine augments adoptive immunotherapy in a murine tumor model.

Recent studies suggest that immunotherapy targeting specific tumor-associated antigens (TAAs) may be beneficial in cancer patients. However, most of these TAAs are tumor type specific and heterogeneous among patients, thus limiting their applications. Here, we describe the de novo induction of a cancer/testis antigen (CTA) for immunotherapy of tumors of various histologies. The murine CTA P1A, normally expressed only in a few tumor lines, could be induced de novo in all P1A-negative cancer lines of eight histologic origins in vitro and in various murine xenografts by systemic administration of 5-aza-2'-deoxycytidine. The induction of P1A expression correlated strongly with demethylation of the CpG island in the promoter region of this gene. The induced antigen was processed and presented properly for recognition by H-2L(d)-restricted P1A-specific CTLs. The combination of a demethylating agent and adoptive transfer of P1A-specific CTL effectively treated lung metastases in syngeneic mice challenged with P1A-negative 4T1 mammary carcinoma cells. These data show a novel strategy of combined chemoimmunotherapy of cancer targeting a CTA induced de novo in a broad range of tumor histologies, and support further evaluation of chromatin-remodeling agents for human cancer therapy.

Modifying the cancer-immune set point using vaccinia virus expressing re-designed interleukin-2.

The complex immune tumour microenvironment requires an equally complex immunotherapy approach, especially when the cancer-immune set point is non-inflamed. Oncolytic viruses expressing immune activating cytokines might optimally modify the immune microenvironment and improve the antitumour effects. In this study, we have explored a variety of IL-2 constructs expressed by a tumour-selective oncolytic vaccinia virus, designed to maintain IL-2 in the tumour microenvironment to reduce systemic toxicity. An IL-2 construct combining a glycosylphosphatidylinositol (GPI) anchor with a rigid peptide linker leads to functional IL-2 expression on the tumour cell surface and in the tumour microenvironment. This virus construct effectively modifies the cancer-immune set point and treats a variety of murine tumour models with no toxic side effects. In combination with PD-1/PD-L1 blockade this virus cures most of the mice with a high tumour burden. This combination represents a treatment for cancers which are to date unresponsive to immunotherapy.

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.

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.

Intravenous and isolated limb perfusion delivery of wild type and a tumor-selective replicating mutant vaccinia virus in nonhuman primates.

In this study we examine the safety, feasibility, and biodistribution of a tumor-selective mutant vaccinia (vvDD) and wild-type WR (vF13) vaccinia after delivery via intradermal or intravenous infection or isolated limb perfusion (ILP) in rhesus macaques. By intradermal inoculation, 10(6) PFU of vvDD caused a minimal skin reaction whereas vF13 caused marked erythema and necrosis with a peak indurated area of 108 cm2. By intravenous delivery, vvDD caused no clinical symptoms of viremia and no viral recovery from tissues, serum, saliva, urine, or feces. In contrast, vF13 caused symptoms of lethargy, anorexia, fever, and signs of viremia. Delivery of vF13 via ILP resulted in numerous cutaneous pox lesions localized solely to the perfused limb with high viral recovery in the perfused skin and muscle. ILP with vvDD resulted in no visible pox lesions and no clinical signs or symptoms of viremia. No long-term toxicity was identified after ILP with 10(9) PFU of vvDD, and no virus was recovered from any tissue, serum, saliva, urine, or fecal sample. These results suggest that vvDD appears to be safe in primates, and thus vvDD should be further investigated for clinical trial in human cancer patients.

Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228.

BACKGROUND: Histone deacetylases (HDACs) modulate chromatin structure by regulating acetylation of core histone proteins. HDAC inhibitors, such as depsipeptide FR901228 (FK228), induce growth arrest and apoptosis in a variety of human cancer cells by mechanisms that cannot be attributed solely to histone acetylation. This study evaluated the mechanisms by which FK228 mediates apoptosis in non-small-cell lung cancer (NSCLC) cells. METHODS: Proliferation and apoptosis were assessed in a panel of NSCLC cell lines that vary in the expression of the growth-regulating proteins p53, pRb, and K-Ras treated with a clinically relevant dose of FK228 (25 ng/mL). Western blot and immunoprecipitation techniques were used to analyze expression of cell-cycle proteins (cyclin A, cyclin E, p53, and p21), signaling-related proteins (ErbB1, ErbB2, and Raf-1), activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2), binding of mutant p53 and Raf-1 to heat shock protein (Hsp)90, and acetylation of Hsp90. RESULTS: FK228 treatment inhibited growth and induced apoptosis in NSCLC cells expressing wild-type or mutant p53. FK228 treatment led to altered expression of cyclin A, cyclin E, and p21, and to reduced expression of mutant, but not wild-type, p53. FK228-treated cells also were depleted of ErbB1, ErbB2, and Raf-1 proteins, and exhibited lower ERK1/2 activity. FK228 treatment also inhibited the binding of mutant p53 and Raf-1 to Hsp90; this inhibition was associated with acetylation of Hsp90. CONCLUSIONS: FK228 depletes the levels of several oncoproteins that are normally stabilized by binding to Hsp90 in cancer cells. The resulting ability of FK228 to diminish signal transduction via pathways involving Raf-1 and ERK may contribute to the potency and specificity of this novel antitumor agent.

CXCL11-Armed oncolytic poxvirus elicits potent antitumor immunity and shows enhanced therapeutic efficacy.

We have armed a tumor-selective oncolytic vaccinia virus (vvDD) with the chemokine (CK) CXCL11, in order to enhance its ability to attract CXCR3+ antitumor CTLs and possibly NK cells to the tumor microenvironment (TME) and improve its therapeutic efficacy. As expected, vvDD-CXCL11 attracted high numbers of tumor-specific T cells to the TME in a murine AB12 mesothelioma model. Intratumoral virus-directed CXCL11 expression enhanced local numbers of CD8+ CTLs and levels of granzyme B, while reducing expression of several suppressive molecules, TGF-ß, COX2, and CCL22 in the TME. Unexpectedly, we observed that vvDD-CXCL11, but not parental vvDD, induced a systemic increase in tumor-specific IFNγ-producing CD8+ T cells in the spleen and other lymph organs, indicating the induction of systemic antitumor immunity. This effect was associated with enhanced therapeutic efficacy and a survival benefit in tumor-bearing mice treated with vvDD-CXCL11, mediated by CD8+ T cells and IFNγ, but not CD4+ T cells. These results demonstrate that intratumoral expression of CXCL11, in addition to promoting local trafficking of T cells and to a lesser extent NK cells, has a novel function as a factor eliciting systemic immunity to cancer-associated antigens. Our data provide a rationale for expressing CXCL11 to enhance the therapeutic efficacy of oncolytic viruses (OVs) and cancer vaccines.

Vaccinia as a vector for gene delivery.

Gene therapy is a promising approach, yet so far it has shown limited effectiveness in many clinical trials, mainly due to insufficient gene transduction. Recombinant vaccinia virus (rVV) has been well developed as a gene delivery vector, initially for protein expression in mammalian cells. rVV has been further developed to express antigens in vivo in generating immunity for protection against specific infectious diseases and cancer. rVVs, as non-replicating viral vectors, have been demonstrated for their great potential as vaccines, for their diminished cytopathic effects, high levels of protein expression and strong immunogenicity, and they are relatively safe in animals and in human patients. A number of clinical trials using rVVs as vaccines have shown promising results for treating infectious diseases and cancer. In the last few years, due to its exceptional ability to replicate in tumour cells, the Western Reserve strain vaccinia has been explored as a replicating oncolytic virus for cancer virotherapy. As more is learned about the functions of viral gene products in controlling the mammalian cell cycle and in disabling cellular defence mechanisms, specific viral functions can be augmented or eliminated to enhance antitumour efficacy and improve tumour cell targeting. General mechanisms by which this oncolytic virus achieves the antitumour efficacy and specificity are reviewed. Specifically, the deletion of the viral genes for thymidine kinase and vaccinia growth factor resulted in a vaccinia mutant with enhanced tumour targeting activity and fully retaining its efficiency of replication in cancer cells. Other potential strategies for improving this vector for gene delivery will also be discussed in this review.

Life after death: targeting high mobility group box 1 in emergent cancer therapies.

High mobility group box 1 (HMGB1), an evolutionarily highly conserved and abundant nuclear protein also has roles within the cytoplasm and as an extracellular damage-associated molecular pattern (DAMP) molecule. Extracellular HMGB1 is the prototypic endogenous 'danger signal' that triggers inflammation and immunity. Recent findings suggest that posttranslational modifications dictate the cellular localization and secretion of HMGB1. HMGB1 is actively secreted from immune cells and stressed cancer cells, or passively released from necrotic cells. During cancer development or administration of therapeutic agents including chemotherapy, radiation, epigenetic drugs, oncolytic viruses, or immunotherapy, the released HMGB1 may either promote or limit cancer growth, depending on the state of progression and vascularization of the tumor. Extracellular HMGB1 enhances autophagy and promotes persistence of surviving cancer cells following initial activation. When oxidized, it chronically suppresses the immune system to promote cancer growth and progression, thereby enhancing resistance to cancer therapeutics. In its reduced form, it can facilitate and elicit innate and adaptive anti-tumor immunity, recruiting and activating immune cells, in conjunction with cytotoxic agents, particularly in early transplantable tumor models. We hypothesize that HMGB1 also functions as an epigenetic modifier, mainly through regulation of NF-kB-dependent signaling pathways, to modulate the behavior of surviving cancer cells as well as the immune cells found within the tumor microenvironment. This has significant implications for developing novel cancer therapeutics.