A Systematic Literature Review of Injection Site Pain Perception in Adult Patients Treated with Citrate-Free and Citrate-Containing Biologic Agents.

OBJECTIVE: To investigate injection site pain (ISP) and other injection site outcomes caused by biologics administered alongside citrate-free (CF) and citrate-containing (CC) formulations. METHODS: Electronic literature databases (Medline, Embase, and Cochrane Library) were systematically searched for clinical trials and observational studies reporting on injection site outcomes after subcutaneous administration of biologics. Studies with unknown excipient formulations were excluded. The primary outcome was ISP, and secondary outcomes included any other reported injection site reactions (ISRs). Meta-analysis approaches were used to aggregate evidence identified via the conducted systematic literature review. RESULTS: A total of two observational studies, two cross-over/sequential trials, and three head-tohead comparison trials directly comparing CF with CC biologics were identified, as well as seven placebo-controlled trials. Evidence from five of the seven direct comparison studies suggested reduced pain perception at the injection site when CF formulations were applied. Findings for other ISRs were balanced between both formulations, with slightly favorable results for preparations without citrate. A meta-analysis of placebo-controlled trials found no significant difference between arms with CF formulations and placebo regarding the proportion of patients experiencing ISP (OR 0.62, 95% CI 0.30-1.28). CONCLUSION: Excipient formulations are rarely specified in studies assessing pain and other ISRs of subcutaneously administered biologics. The available data indicate that subcutaneous administration of biologic agents without citrate may be associated with lower pain perception outcomes compared with treatment using CC formulations. Importantly, ISP is influenced by many factors which may have affected the results. More research is needed to assess how formulation excipients influence ISRs.

Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers.

Vesicular stomatitis virus (VSV) represents an attractive oncolytic virotherapy platform because of its potent tumor cell-killing and immune-stimulating properties; yet the clinical translation of VSV faces numerous challenges, such as inefficient systemic delivery and severe side effects such as neurotoxicity. We hypothesized that we could overcome these limitations and simultaneously enhance the therapy, by combining VSV with adoptively transferred T cell receptor (TCR) transgenic T cells as carrier cells. We show that CD8+ T central memory cells (CD8+ T cm) can be efficiently loaded with VSV, they support intracellular virus production, and they can efficiently transfer VSV to tumor cells without compromising their own viability or antitumor reactivity. Loading VSV onto CD8+ T cm not only improves the safety compared with systemic administration of naked virus, but this approach also allows for an effective delivery of virus to its tumor target, resulting in an effective combination therapy in NSG mice bearing subcutaneous human acute myeloid leukemia (AML) tumors. We conclude that the combination of potent tumor debulking provided by the oncolytic VSV with the added effector functions afforded by the cytotoxic immune carrier cells results in a potent and safer immunotherapeutic, which can be further developed for clinical translation.

A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma.

Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named recombinant VSV-NDV (rVSV-NDV), wherein the VSV backbone is conserved but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. In comparison to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytium formation, allowing efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wild-type VSV, resulting in a markedly enhanced safety profile, even in immune-deficient NOD.CB17-prkdcscid/NCrCrl (NOD-SCID) mice, which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic hepatocellular carcinoma (HCC)-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus without risking the safety and environmental threats associated with either parental vector. Taking the data together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus.IMPORTANCE The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Randomized phase II placebo controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma.

BACKGROUND & AIMS: Codrituzumab, a humanized monoclonal antibody against Glypican-3 (GPC3) that is expressed in hepatocellular carcinoma (HCC), interacts with CD16/FcγRIIIa and triggers antibody-dependent cytotoxicity. Codrituzumab was studied vs. placebo in a randomized phase II trial in advanced HCC patients who had failed prior systemic therapy. METHODS: Patients with advanced HCC who had failed prior systemic therapy, ⩾18years, Eastern cooperative oncology group (ECOG) 0-1, Child-Pugh A were randomized 2:1 to biweekly codrituzumab 1600mg vs. placebo. Patients were stratified based on GPC3 immunohistochemical expression: 2+/3+, 1+, and 0. Primary endpoint was progression free survival. Secondary endpoints include overall survival (OS), tolerability, pharmacokinetics, and an exploratory endpoint in biomarkers analysis. RESULTS: 185 patients were enrolled: 125 received codrituzumab and 60 placebo: Median age 64/63, 85/75% male, 46/42% Asian, ECOG 0 65/63%, 74/77% having vascular invasion and/or extra-hepatic metastasis. 84%/70% had prior sorafenib. Drug exposure was 98.4% of planned dose, with an identical adverse events profile between the 2 groups. The median progression free survival and overall survival in the codrituzumab vs. placebo groups in months were: 2.6 vs. 1.5 (hazard ratios 0.97, p=0.87), and 8.7 vs. 10 (hazard ratios 0.96, p=0.82). Projected Ctrough at cycle 3day 1 based exposure, high CD16/FcγRIIIa on peripheral immune cells, and GPC3 expression in the tumor, were all associated with prolonged progression free survival and overall survival. CONCLUSIONS: Codrituzumab did not show clinical benefit in this previously treated HCC population. Whether higher codrituzumab drug exposure or the use of CD16 and GPC3 as potential biomarkers would improve outcome remain unanswered questions. LAY SUMMARY: Codrituzumab is a manufactured antibody against a liver cancer protein called glypican-3. In this clinical trial, codrituzumab was not found be effective against liver cancer. It was suggested though that a higher dose of codrituzumab or selecting patients with high level of glypican-3 or its mediator CD16 might improve outcome. CLINICAL TRIAL REGISTRATION: This trial is registered at Clinicaltrials.gov (NCT01507168).

Influence of Sorafenib and Bevacizumab on pancreatic volume - A monocentric CT based analysis.

BACKGROUND/OBJECTIVES: Angiogenesis plays a central role in tumor growth and metastasis and tyrosine kinases are crucial in the modulation of growth factor signaling. Several side effects of tyrosine kinase inhibitors have been reported, including diarrhea due to pancreatic insufficiency. The suspected mechanism is the anti-angiogenetic effect of the inhibited vascular endothelial growth factor (VEGF) causing a disturbance of the microvasculation. The aim of the present study was to determine the volume of the pancreas before and after a therapy both with the multi-tyrosine kinase inhibitor Sorafenib and Bevacizumab, which is a humanized monoclonal immunoglobulin G1 antibody against VEGF. METHODS: Retrospective monocentric study including 42 patients who received either Sorafenib, Bevacizumab combined with Flourouracil and/or Irinotecan, or singly Flourouracil and Irinotecan for different non-pancreatic malignancies. The volume of the pancreas was measured before and after therapy by CT-scan based volumetry. RESULTS: The pancreatic volume was statistically significantly lower after treatment with Sorafenib (75.4 mL vs. 71.0 mL; p = 0.006) or Bevacizumab and Fluorouracil ± Irinotecan (71.8 mL vs. 62.6 mL; p = 0.020). The pancreatic volume did not change statistically significantly after treatment with Fluorouracil ± Irinotecan only (51.1 mL vs. 49.9 mL; p = 0.142). CONCLUSIONS: Pancreatic volume decreases statistically significantly under treatment with both the multi-tyrosine kinase inhibitor Sorafenib and the angiogenesis inhibitor Bevacizumab. This volume reduction is most likely due to a reduced microvasculation by inhibition of VEGF.

Model Matters: Differences in Orthotopic Rat Hepatocellular Carcinoma Physiology Determine Therapy Response to Sorafenib.

PURPOSE: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. EXPERIMENTAL DESIGN: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. RESULTS: Histologically and biochemically confirmed liver damage resulted in increased (18)F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1-3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. CONCLUSIONS: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies.

Characterization of magnetic viral complexes for targeted delivery in oncology.

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2(*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2(*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

PET imaging of oncolytic VSV expressing the mutant HSV-1 thymidine kinase transgene in a preclinical HCC rat model.

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Sorting Out Pandora's Box: Discerning the Dynamic Roles of Liver Microenvironment in Oncolytic Virus Therapy for Hepatocellular Carcinoma.

Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome.

Antifibrotic properties of transarterial oncolytic VSV therapy for hepatocellular carcinoma in rats with thioacetamide-induced liver fibrosis.

Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Depletion of the transcriptional coactivators megakaryoblastic leukaemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence.

Megakaryoblastic leukaemia 1 and 2 (MKL1/2) are coactivators of the transcription factor serum response factor (SRF). Here, we provide evidence that depletion of MKL1 and 2 abolishes hepatocellular carcinoma (HCC) xenograft growth. Loss of the tumour suppressor deleted in liver cancer 1 (DLC1) and the subsequent activation of RhoA were prerequisites for MKL1/2 knockdown-mediated growth arrest. We identified oncogene-induced senescence as the molecular mechanism underlying the anti-proliferative effect of MKL1/2 knockdown. MKL1/2 depletion resulted in Ras activation, elevated p16 expression and hypophosphorylation of the retinoblastoma (Rb) protein in DLC1-deficient HCC cells. Interestingly, reconstitution of HuH7 HCC cells with DLC1 also induced senescence. Evaluation of the therapeutic efficacy of MKL1/2 knockdown in vivo revealed that systemic treatment of nude mice bearing HuH7 tumour xenografts with MKL1/2 siRNAs complexed with polyethylenimine (PEI) completely abolished tumour growth. The regression of the xenografts was associated with senescence. Importantly, PEI-complexed MKL1 siRNA alone was sufficient for complete abrogation of HCC xenograft growth. Thus, MKL1/2 represent promising novel therapeutic targets for the treatment of HCCs characterized by DLC1 loss.

The potential of recombinant vesicular stomatitis virus-mediated virotherapy against metastatic colon cancer.

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related mortality in the United States. The liver and lung are the most common sites of distant metastasis of CRC. The approval of newer chemotherapeutic agents such as oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab has significantly improved survival, yet the majority of patients still succumb to the disease in less than 2 years. Novel therapeutic agents that can provide significant clinical benefit for metastatic CRC patients are needed. Oncolytic vesicular stomatitis virus (VSV) is a promising tool as a cancer therapeutic agent. In this study, we examined the feasibility of repeated intravenous infusions of rVSV in multiple CRC lung metastases, compared with repeated hepatic arterial administration in multifocal CRC liver metastasis in immune competent rats. We established a multifocal liver metastases model or the multiple lung metastases model using a CRC cell line, RCN-H4, implanted into syngeneic F344/DuCrj rats. 4.0x10(6) plaque-forming units (pfu) of recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein [rVSV-NDV/F(L289A)] were administered 3 times for 3 consecutive days locally via the hepatic artery for liver metastases or systemically via the penial vein for lung metastases. In the liver metastasis model, significantly enhanced survival was observed with rVSV-NDV/F(L289A)-treated rats (P=0.0196). Median survival was 110 and 25 days, respectively. In addition, 4 out of 7 of the rVSV-NDV/F(L289A)-treated rats demonstrated long-term survival exceeding 100 days. The long-term surviving rats were sacrificed to evaluate for residual malignancy. Liver tumors were not detected. In the lung metastasis model, median survival was 10 [VSV-NDV/F(L289A)-treated rats] and 7 days (control). Although survival was significantly prolonged (P<0.001), none of the rats achieved long-term survival. VSV virotherapy has potential for CRC liver and lung metastases, although systemic venous delivery is much less effective than locoregional delivery such as hepatic arterial infusion.

Posttranslational modification of vesicular stomatitis virus glycoprotein, but not JNK inhibition, is the antiviral mechanism of SP600125.

Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogen-activated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/ß). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein.

Replicating viral vectors for cancer therapy: strategies to synergize with host immune responses.

Tumour-specific replicating (oncolytic) viruses are novel anticancer agents, currently under intense investigation in preclinical studies and phase I-III clinical trials. Until recently, most studies have focused on the direct antitumour properties of these viruses. There is now an increasing body of evidence indicating that host immune responses may be critical to the efficacy of oncolytic virotherapy. Although the immune response to oncolytic viruses can rapidly restrict viral replication, thereby limiting the efficacy of therapy, oncolytic virotherapy also has the potential to induce potent antitumoural immune effectors that destroy those cancer cells, which are not directly lysed by virus. In this review, we discuss the role of the immune system in terms of antiviral and antitumoural responses, as well as strategies to evade or promote these responses in favour of improved therapeutic potentials.