Ghassan Alusi

Lister strain of vaccinia virus armed with endostatin–angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer

Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin–angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin–angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin–angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.

A Novel Therapeutic Regimen to Eradicate Established Solid Tumors with an Effective Induction of Tumor-Specific Immunity

Purpose: The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses. Experimental Design: The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion. Results: The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses. Conclusion: These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role. Clin Cancer Res; 18(24); 6679–89. ©2012 AACR.

Combined blockade of signalling pathways shows marked anti-tumour potential in phaeochromocytoma cell lines

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.

Lister Vaccine Strain of Vaccinia Virus Armed with the Endostatin–Angiostatin Fusion Gene: An Oncolytic Virus Superior to dl1520 (ONYX-015) for Human Head and Neck Cancer

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

Vaccinia virus, a promising new therapeutic agent for pancreatic cancer

The poor prognosis of pancreatic cancer patients signifies a need for radically new therapeutic strategies. Tumor-targeted oncolytic viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumor cells as well as induce antitumor effects by multiple action mechanisms. Vaccinia virus has several inherent features that make it particularly suitable for use as an oncolytic agent. In this review, we will discuss the potential of vaccinia virus in the management of pancreatic cancer in light of our increased understanding of cellular and immunological mechanisms involved in the disease process as well as our extending knowledge in the biology of vaccinia virus.

Lister strain vaccinia virus with thymidine kinase gene deletion is a tractable platform for development of a new generation of oncolytic virus

Vaccinia virus (VV) has many attractive characteristics as a potential cancer therapeutic. There are several strains of VV. The nonvaccine strain Western Reserve VV with deletion of both the thymidine kinase and the viral growth factor genes (known as WRDD) has been reported as the most potent tumor-targeted oncolytic VV. Other strains, such as the European vaccine Lister strain, are largely untested. This study evaluated the antitumor potency and biodistribution of different VV strains using in vitro and in vivo models of cancer. Lister strain virus with thymidine kinase gene deletion (VVΔTK) demonstrated superior antitumor potency and cancer-selective replication in vitro and in vivo, compared with WRDD, especially in human cancer cell lines and immune-competent hosts. Further investigation of functional mechanisms revealed that Lister VVΔTK presented favorable viral biodistribution within the tumors, with lower levels of proinflammatory cytokines compared with WRDD, suggesting that Lister strain may induce a diminished host inflammatory response. This study indicates that the Lister strain VVΔTK may be a particularly promising VV strain for the development of the next generation of tumor-targeted oncolytic therapeutics.

Characterization of SNARE Proteins in Human Pituitary Adenomas: Targeted Secretion Inhibitors as a New Strategy for the Treatment of Acromegaly?

CONTEXT Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. OBJECTIVE Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. DESIGN We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. SETTING The study was conducted in University Hospitals. PATIENTS We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. OUTCOME Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. RESULTS SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. CONCLUSIONS SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.

Gene therapy and nasopharyngeal carcinoma.

In 2003, a non-replicating adenoviral gene therapy product received the world`s first government licence for the treatment of head and neck cancer. Two years later approval was granted to a replication-selective adenovirus for the treatment of nasopharyngeal carcinoma in combination with chemotherapy. This review introduces the reader to gene therapy as an emerging treatment modality, and outlines its application to the management of nasopharyngeal carcinoma by examining recent pre-clinical and clinical research.

Viral gene therapy for head and neck cancer.

BACKGROUND Viral gene therapy is a promising new treatment modality for head and neck cancer. This paper provides the reader with a review of the relevant literature in this field. RESULTS There are government licensed viral gene therapy products currently in use for head and neck cancer, utilised in conjunction with established treatment modalities. The viruses target tumour-associated genes, with the first licensed virus replacing p53 gene function, which is frequently lost in tumourigenesis. Oncolytic viruses selectively destroy cancer cells through viral replication and can be armed with therapeutic transgenes. CONCLUSION Despite considerable advances in this field over the last 40 years, further research is needed to improve the overall efficacy of the viruses and allow their widespread utilisation in the management of head and neck cancer.

649. Targeting Innate Host Immunity Through PI3K Delta for Enhancement of Systemic Delivery of Oncolytic Vaccinia Virus

Oncolytic vaccinia virus (VV) has shown promise for the delivery of gene therapy and has the potential to be delivered systemically to treat metastatic cancer. VV is known to induce a strong host immune response, which requires immunocompetent models for evaluation. We investigated the systemic delivery of oncolytic vaccinia virus to murine tumours in immunocompetent mice using tumour-targeted oncolytic vaccinia virus and found that VV was unable to efficiently infect tumours due to clearance, mostly by splenic macrophages. Depletion of macrophages using clodronate liposomes (CL) facilitated the systemic delivery of vaccinia virus to subcutaneous, established murine tumours. However CL non-selectively deplete macrophages and potentially diminish any beneficial macrocytic activity in the tumour microenvironment unrelated to viral clearance. Consequently, a more appropriate agent is needed.Macrophages recognise and ingest pathogenic microorganisms through phagocytosis, a process for which several lines of evidence have highlighted an important role for phosphatidylinositol 3-kinases. Thus, PI3K inhibitors could potentially enhance systemic delivery of VV. Eight mammalian isoforms of PI3K exist, but the specific isoforms involved in macrophage phagocytosis of VV have not been elucidated. In this study, we, for the first time, demonstrated that PI3K delta is a major player affecting monocytes to clear vaccine virus. IC87114 (p110 delta inhibitor) is effective at reducing uptake of VV by macrophages. Subsequently, it was confirmed that IC87114 affects attachment of the virus to macrophages but plays no role in internalisation. Furthermore IC87114 is neither directly toxic to a panel of cancer cells nor oncolytically additive nor synergetic when combined with VV in vitro. Biodistribution studies have established that IC87114 combined with VV results in statistically more virus detected in tumours compared to the VV alone treated groups with similar limited off target effects. Finally three different efficacy studies have demonstrated statistically significant superior tumour response in IC87114 group. In conclusion, p110 delta blockade is an effective strategy for enhancing systemic deliver of VV in a pre-clinical model and could be a useful adjuvant in VV clinical trials