Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

A capsid-modified, conditionally replicating oncolytic adenovirus vector expressing TRAIL Leads to enhanced cancer cell killing in human glioblastoma models

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, and patients rarely survive for more than 2 years. Gene therapy may offer new treatment options and improve the prognosis for patients with GBM. Adenovirus-mediated gene therapy strategies for brain tumors have been limited by inefficient gene transfer due to low expression of the adenovirus serotype 5 (Ad5) receptor. We have used an adenovirus vector that specifically replicates in tumor cells and uses an Ad5 capsid and the adenovirus serotype (Ad35) fiber for efficient infection of malignant tumor cells. This vector also expresses adenovirus E1A and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a tumor-specific manner. Here, we show that this oncolytic vector (Ad5/Ad35.IR-E1A/TRAIL) efficiently infects the GBM tumor cell lines SF767, T98G, and U-87 MG. Tumor cell killing was markedly enhanced with Ad5/Ad35.IR-E1A/TRAIL compared with wild-type Ad5 and Ad35 virus or Ad5/Ad35.IR-E1A- vectors without TRAIL expression in vitro. In vivo experiments using s.c. xenografted U-87 MG cells in NOD/SCID mice showed a significant growth delay of tumors after i.t. injection of Ad5/Ad35.IR-E1A/TRAIL, whereas adenovirus wild-type injections showed only marginal or no effect. Our findings indicate that the use of a capsid-modified adenoviral vector, in combination with TRAIL expression, is a promising novel approach for gene therapy of glioblastoma.     

Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system

Targeting the human papillomavirus (HPV) E7 antigen to dendritic cells with the adenylate cyclase (CyaA) of Bordetella pertussis as a vaccine vector led to potent therapeutic immune responses against TC-1 tumors in a murine model of cervical carcinoma induced by HPV. However, as the time between tumor graft and vaccination increased, the antitumor efficacy of the CyaA-E7 vaccine gradually decreased. The vaccine had no effect if the tumor diameter was >8 mm. Analyses of regulatory cells recruited during TC-1 tumor growth revealed a high number of splenic MDSCs and a large percentage of regulatory T cells, particularly in the tumor. Administration of a tritherapy including CpG complexed with a cationic lipid, low-dose cyclophosphamide, and the CyaA-E7 vaccine completely overcame tumor-associated immunosuppression and eradicated large, established tumors in almost all treated animals. This strong antitumor response was followed by a large expansion of regulatory T cells in tumor, spleen, and tumor-draining lymph nodes and of splenic neutrophils. These findings indicate that immunotherapeutic strategies that simultaneously target innate, adaptive, and regulatory components of the immune system are effective in the eradication of large tumors.     

Intratumor injection of oncolytic adenovirus expressing HSP70 prolonged survival in melanoma B16 bearing mice by enhanced immune response

Heat shock proteins (HSPs) possess potent antitumor ability to stimulate immune response. We postulated that intratumor injection of oncolytic adenovirus over-expressing HSPs might be able to exert antitumor activity by inducing antitumor immune response in immunocompetent hosts in addition to the oncolytic activity. In this study, two recombinant oncolytic adenoviruses, Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a, were constructed with or without the HSP expression cassette. The HSP expression and cytopathic killing effect in mouse B16 melanoma and human PLC/PRF/5 hepatoma cells were measured after in vitro infection of adenoviruses. Survival rate of immunocompetent C57/BL mice was observed following intratumor injection of recombinant adenoviruses in B16 melanoma xenograft models. To detect the evidence of immune responses, hematoxylin and eosin staining and RT-PCR for IFN-gamma expression in tumor tissues were performed. The Ad.CMV.HSP.IRES.E1a induced significantly higher HSP expression level than Ad.CMV.IRES.E1a in both B16 and PLC/ PRF/5 cells. The cytocidal efficacy of Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a in PLC/PRF/5 cells was much higher than that in B16 cells, where the two adenoviruses showed similarly very weak oncolytic effect in vitro. However, Ad.CMV.HSP.IRES. E1a improved animal survival rate and exhibited more potent anti-tumor efficiency than Ad.CMV.IRES.E1a in B16 xenograft models. The enhanced efficacy might be mainly attributed to the HSP-mediated immune activity, as evidenced by the up-regulated expression of IFN-gamma and local heavier intratumor inflammatory cell infiltration. These results indicated that the recombinant oncolytic adenovirus over-expressing HSPs possessed powerful in vivo anti-tumor efficacy and might be a valuable approach for cancer immune gene therapy.     

Anticancer activity of oncolytic adenovirus vector armed with IFN-alpha and ADP is enhanced by pharmacologically controlled expression of TRAIL

We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.     

Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL

Cancer-targeting gene-viro-therapy is a promising cancer therapeutic strategy that strengthens the antitumor effect of oncolytic viruses by expressing an inserted foreign antitumor gene. To achieve liver cancer targeting and to improve the safety of the ZD55 vector (a widely-used E1B55KD gene-deleted oncolytic adenoviral vector (OV), we previously constructed), we designed a novel OV named Ad·AFP·D55 that selectively replicates in hepatocellular carcinoma (HCC) cells by replacing the E1A promoter with the liver-cancer specific α-Fetoprotein (AFP) promoter based on the ZD55 vector. We found that the oncolytic adenoviruses Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL express tumor-suppressor gene interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), respectively, significantly suppressed the HCC cell growth in vitro by inducing apoptosis by the caspase-8 and mitochondria-dependent caspase-9 signaling pathways. Furthermore, the combined treatment of Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL showed strong antitumor effects in vivo by significantly inhibiting the tumor growth in HCC HuH-7 cell xenograft mice, and markedly increasing animal survival rate. Therefore, this novel HCC cell-targeting OV carrying tumor-suppressor genes may provide a promising approach for liver cancer gene therapy.     

Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models

Tumor-associated antigens (TAAs) include overexpressed self-antigens (for example, Her2/neu) and tumor virus antigens (for example, HPV-16 E6/E7). Although in cancer patients, TAA-specific CD4+ and CD8+ cells are often present, they are not able to control tumor growth. In recent studies, it became apparent that tumor site-located immune evasion mechanisms contribute to this phenomenon and that regulatory T cells have a major role. We tested in Her2/neu+ breast cancer and HPV-16 E6/E7+ cervical cancer mouse models, whether intratumoral expression of immunostimulatory proteins (ISPs), for example, recombinant antibodies (αCTLA-4, αCD137, αCD3), cyto/chemokines (IL-15, LIGHT, mda-7) and costimulatory ligands (CD80), through adenovirus(Ad)-mediated gene transfer would overcome resistance. In both the breast and cervical cancer model, none of the Ad.ISP vectors displayed a significant therapeutic effect when compared with an Ad vector that lacked a transgene (Ad.zero). However, the combination of Ad.ISP vectors with systemic T regulatory (Treg) depletion, using anti-CD25?mAb (breast cancer model) or low-dose cyclophosphamide (cervical cancer model) resulted in a significant delay of tumor growth in mice treated with Ad.αCTLA4. In the cervical cancer model, we also demonstrated the induction of a systemic antitumor immune response that was able to delay the growth of distant tumors. Ad.αCTLA4-mediated tumor-destructive immune responses involved NKT and CD8+ T cells. In both models no autoimmune reactions were observed. This study shows that Ad.αCTLA4 in combination with systemic Treg depletion has potentials in the immunotherapy of cancer.     

Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.     

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.     

Transthyretin-driven oncolytic adenovirus suppresses tumor growth in orthotopic and ascites models of hepatocellular carcinoma

Strategies to increase antitumor efficacy of oncolytic adenoviruses are actively investigated. We have previously shown that E1B-55 kDa-deleted adenovirus, designated Ad5WS1, has therapeutic potential for treating hepatocellular carcinoma (HCC). To achieve HCC-restricted replication of oncolytic adenovirus, we generated Ad5WS2, an E1B-55 kDa-deleted adenovirus with its E1A gene driven by the liver-specific transthyretin promoter. Our results showed that Ad5WS2 could replicate within tumor cells where the transthyretin gene was expressed. Mouse transthyretin promoter was active in murine and human HCC cells, but relatively quiescent in cells of non-liver origin. Ad5WS2 caused severe cytolytic effect on HCC cells, but was much attenuated in non-HCC cells. Peritoneal administration of Ad5WS2 into mice bearing liver tumors grown in ascites resulted in enhanced survival. In an orthotopic HCC model, Ad5WS2, when systemically administered, exerted higher antitumor effects than Ad5WS1. Lack of viral replication in normal organs and minimal hepatic toxicity was noted after Ad5WS2 treatment. Furthermore, the antitumor effect of Ad5WS2 could be enhanced when combined with chemotherapeutic agent cisplatin in the ascites tumor model. These results suggest that E1B-55 kDa-deleted adenovirus driven by the transthyretin promoter may be a safer and more efficacious oncolytic agent for the treatment of primary and metastatic HCC. ( Cancer Sci 2009; 100: 537–545)     

Relaxin-expressing, fiber chimeric oncolytic adenovirus prolongs survival of tumor-bearing mice

Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.     

Intratumoral coadministration of hyaluronidase enzyme and oncolytic adenoviruses enhances virus potency in metastatic tumor models

PURPOSE: Evaluate the codelivery of hyaluronidase enzyme with oncolytic adenoviruses to determine whether it improves the spread of the virus throughout tumors, thereby leading to a greater overall antitumor efficacy in tumor models. EXPERIMENTAL DESIGN: The optimal dose of hyaluronidase that provided best transduction efficiency and spread of a green fluorescent protein (GFP)-expressing adenovirus within tumors was combined with oncolytic viruses in tumor models to determine whether the combination treatment results in an improvement of antitumor efficacy. RESULTS: In mice injected with the adenovirus Ad5/35GFP and an optimal dose of hyaluronidase (50 U), a significant increase in the number of GFP-expressing cells was observed when compared with animals injected with virus only (P < 0.0001). When the oncolytic adenoviruses Ad5OV or Ad5/35 OV (OV-5 or OV5T35H) were codelivered with 50 U of hyaluronidase, a significant delay in tumor progression was observed, which translated into a significant increase in the mean survival time of tumor-bearing mice compared with either of the monotherapy-treated groups (P < 0.0001). Furthermore, the mice that received the combination of Ad5/35 OV and hyaluronidase showed the best antitumor efficacy. Importantly, the combination treatment did not increase the metastatic potential of the tumors. Lastly, the increase in virus potency observed in animals injected with both enzyme and virus correlated with enhanced virus spread throughout tumors. CONCLUSION: Antitumor activity and overall survival of mice bearing highly aggressive tumors are significantly improved by codelivery of oncolytic adenoviruses and hyaluronidase when compared with either of the monotherapy-treated groups, and it may prove to be a potent and novel approach to treating patients with cancer.     

Coadministration of a herpes simplex virus-2 based oncolytic virus and cyclophosphamide produces a synergistic antitumor effect and enhances tumor-specific immune responses

Despite their unique property of selective replication and propagation in tumor tissues, oncolytic viruses have had only limited antitumor effects in cancer patients. One of the major reasons is probably the host's immune defense mechanisms, which can restrict the ability of the virus to replicate and spread within tumors. The innate immune system, which can be rapidly activated during virus infection, likely plays a more pivotal antiviral role than does acquired immunity, as the antitumor effect of an oncolytic virus is mainly generated during the acute phase of virus replication. To exploit the potential of cyclophosphamide, a cancer chemotherapeutic drug that also inhibits innate immune responses, to enhance the activity of oncolytic viruses, we evaluated the effect of coadministration of this drug with a herpes simplex virus-2-based oncolytic virus (FusOn-H2) against Lewis lung carcinoma, which is only semipermissive to infection with FusOn-H2. This strategy synergistically enhanced the antitumor effect against lung carcinoma growing in mice. It also potentiated the ability of FusOn-H2 to induce tumor-specific immune responses. Together, our results suggest that coadministration of FusOn-H2 with cyclophosphamide would be a feasible way to enhance the antitumor effects of this oncolytic virus in future clinical trials.     

Secretable chaperone Grp170 enhances therapeutic activity of a novel tumor suppressor, mda-7/IL-24

Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) is a cancer-specific, apoptosis-inducing gene with broad-spectrum antitumor activity, making it an ideal candidate for a novel cancer gene therapy. A systemic and sustained antitumor immune response generated at the time of initial molecular-targeted therapy would provide additional clinical benefits in cancer patients, resulting in improved prevention of tumor recurrence. In this study, we explored the therapeutic efficacy of intratumoral delivery of a nonreplicating adenoviral vector encoding mda-7/IL-24 (Ad.mda-7) and a secretable form of endoplasmic reticulum resident chaperone grp170 (Ad.sgrp170), a potent immunostimulatory adjuvant and antigen carrier. Intratumoral administration of Ad.mda-7 in combination with Ad.sgrp170 was more effective in controlling growth of TRAMP-C2 prostate tumor compared with either Ad.mda-7 or Ad.sgrp170 treatment. Generation of systemic antitumor immunity was shown by enhanced protection against subsequent tumor challenge and improved control of distant tumors. The combined treatments enhanced antigen and tumor-specific T-cell response, as indicated by increased IFN-gamma production and cytolytic activity. Antibody depletion suggests that CD8(+) T cells may be involved in the antitumor effect of the dual molecule-targeted therapies. Therefore, introducing immunostimulatory chaperone grp170 in situ strongly promotes the "immunogenic" cell death when delivered to the mda-7/IL-24-induced apoptotic tumor cells, indicating that an improved anticancer efficacy may be achieved by concurrently targeting both tumor and immune compartments. Given multiple undefined antigens present endogenously within prostate cancer, these data provide a rationale for combining sgrp170-based vaccine strategy with mda-7/IL-24-targeted cancer therapy to induce durable systemic immunity.     

Human dendritic cells efficiently phagocytose adenoviral oncolysate but require additional stimulation to mature

Oncolytic adenoviruses are emerging agents for treatment of cancer by tumor-restricted virus infection and cell lysis. Clinical trials have shown that oncolytic adenoviruses are well tolerated in patients but also that their antitumor activity needs improvement. A promising strategy toward this end is to trigger systemic and prolonged antitumor immunity by adenoviral oncolysis. Antitumor immune activation depends in large part on antigen presentation and T cell activation by dendritic cells (DCs). Thus, it is likely that the interaction of lysed tumor cells with DCs is a key determinant of such "oncolytic vaccination." Our study reveals that human DCs effectively phagocytose melanoma cells at late stages of oncolytic adenovirus infection, when the cells die showing preferentially features of necrotic cell death. Maturation, migration toward CCL19 and T cell stimulatory capacity of DCs, crucial steps for immune induction, were, however, not induced by phagocytosis of oncolysate, but could be triggered by a cytokine maturation cocktail. Therefore, oncolytic adenoviruses and adenoviral oncolysate did not block DC maturation, which is in contrast to reports for other oncolytic viruses. These results represent a rationale for inserting immunostimulatory genes into oncolytic adenovirus genomes to assure critical DC maturation. Indeed, we report here that adenoviral transduction of melanoma cells with CD40L during oncolysis triggers the maturation of human DCs with T cell stimulatory capacity similar to DCs matured by cytokines. We conclude that triggering and shaping DC-induced antitumor immunity by oncolytic adenoviruses "armed" with immunostimulatory genes holds promise for improving the therapeutic outcome of viral oncolysis in patients.     

表达LAG-3抗体的溶瘤腺病毒对成胶质细胞瘤的抗瘤性

目的:探讨共表达淋巴细胞活化基因3(lymphocyte activation gene 3,LAG-3)抗体(LAG-3 antibody,aLAG)的溶瘤腺病毒对成胶质细胞瘤的抗肿瘤活性.方法:在溶瘤腺病毒Ad3的骨架中插入aLAG序列,获得重组溶瘤腺病毒Ad3-aLAG.应用WB法检测感染成胶质细胞瘤GL261细...