AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Recent Advancements in AAV-Vectored Immunoprophylaxis in the Nonhuman Primate Model.

Monoclonal antibodies (mAbs) are important treatment modalities for preventing and treating infectious diseases, especially for those lacking prophylactic vaccines or effective therapies. Recent advances in mAb gene cloning from naturally infected or immunized individuals has led to the development of highly potent human mAbs against a wide range of human and animal pathogens. While effective, the serum half-lives of mAbs are quite variable, with single administrations usually resulting in short-term protection, requiring repeated doses to maintain therapeutic concentrations for extended periods of time. Moreover, due to their limited time in circulation, mAb therapies are rarely given prophylactically; instead, they are generally administered therapeutically after the onset of symptoms, thus preventing mortality, but not morbidity. Adeno-associated virus (AAV) vectors have an established record of high-efficiency in vivo gene transfer in a variety of animal models and humans. When delivered to post-mitotic tissues such as skeletal muscle, brain, and heart, or to organs in which cells turn over slowly, such as the liver and lungs, AAV vector genomes assume the form of episomal concatemers that direct transgene expression, often for the lifetime of the cell. Based on these attributes, many research groups have explored AAV-vectored delivery of highly potent mAb genes as a strategy to enable long-term expression of therapeutic mAbs directly in vivo following intramuscular or intranasal administration. However, clinical trials in humans and studies in nonhuman primates (NHPs) indicate that while AAVs are a powerful and promising platform for vectored immunoprophylaxis (VIP), further optimization is needed to decrease anti-drug antibody (ADA) and anti-capsid antibody responses, ultimately leading to increased serum transgene expression levels and improved therapeutic efficacy. The following review will summarize the current landscape of AAV VIP in NHP models, with an emphasis on vector and transgene design as well as general delivery system optimization. In addition, major obstacles to AAV VIP, along with implications for clinical translation, will be discussed.

AAV-Vectored Expression of Marburg Virus-Neutralizing Antibody MR191 Provides Complete Protection From Challenge in a Guinea Pig Model.

Although there are no approved countermeasures available to prevent or treat disease caused by Marburg virus (MARV), potently neutralizing monoclonal antibodies (mAbs) derived from B cells of human survivors have been identified. One such mAb, MR191, has been shown to provide complete protection against MARV in nonhuman primates. We previously demonstrated that prophylactic administration of an adeno-associated virus (AAV) expressing MR191 protected mice from MARV. Here, we modified the AAV-MR191 coding sequence to enhance efficacy and reevaluated protection in a guinea pig model. Remarkably, 4 different variants of AAV-MR191 provided complete protection against MARV, despite administration 90 days prior to challenge. Based on superior expression kinetics, AAV-MR191-io2, was selected for evaluation in a dose-reduction experiment. The highest dose provided 100% protection, while a lower dose provided ∼88% protection. These data confirm the efficacy of AAV-mediated expression of MR191 and support the further development of this promising MARV countermeasure.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge.

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 µg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

Overcoming Barriers to Preventing and Treating P. aeruginosa Infections Using AAV Vectored Immunoprophylaxis.

Pseudomonas aeruginosa is a bacterial pathogen of global concern and is responsible for 10-15% of nosocomial infections worldwide. This opportunistic bacterial pathogen is known to cause serious complications in immunocompromised patients and is notably the leading cause of morbidity and mortality in patients suffering from cystic fibrosis. Currently, the only line of defense against P. aeruginosa infections is antibiotic treatment. Due to the acquired and adaptive resistance mechanisms of this pathogen, the prevalence of multidrug resistant P. aeruginosa strains has increased, presenting a major problem in healthcare settings. To date, there are no approved licensed vaccines to protect against P. aeruginosa infections, prompting the urgent need alternative treatment options. An alternative to traditional vaccines is vectored immunoprophylaxis (VIP), which utilizes a safe and effective adeno-associated virus (AAV) gene therapy vector to produce sustained levels of therapeutic monoclonal antibodies (mAbs) in vivo from a single intramuscular injection. In this review, we will provide an overview of P. aeruginosa biology and key mechanisms of pathogenesis, discuss current and emerging treatment strategies for P. aeruginosa infections and highlight AAV-VIP as a promising novel therapeutic platform.

Adeno-associated virus mediated expression of monoclonal antibody MR191 protects mice against Marburg virus and provides long-term expression in sheep.

Vectored monoclonal antibody (mAb) expression mediated by adeno-associated virus (AAV) gene delivery leads to sustained therapeutic mAb expression and protection against a wide range of infectious diseases in both small and large animal models, including nonhuman primates. Using our rationally engineered AAV6 triple mutant capsid, termed AAV6.2FF, we demonstrate rapid and robust expression of two potent human antibodies against Marburg virus, MR78 and MR191, following intramuscular (IM) administration. IM injection of mice with 1 × 1011 vector genomes (vg) of AAV6.2FF-MR78 and AAV6.2FF-MR191 resulted in serum concentrations of approximately 141 µg/mL and 195 µg/mL of human IgG, respectively, within the first four weeks. Mice receiving 1 × 1011 vg (high) and 1 × 1010 vg (medium) doses of AAV6.2FF-MR191 were completely protected against lethal Marburg virus challenge. No sex-based differences in serum human IgG concentrations were observed; however, administering the AAV-mAb over multiple injection sites significantly increased serum human IgG concentrations. IM administration of three two-week-old lambs with 5 × 1012 vg/kg of AAV6.2FF-MR191 resulted in serum human IgG expression that was sustained for more than 460 days, concomitant with low levels of anti-capsid and anti-drug antibodies. AAV-mAb expression is a viable method for prolonging the therapeutic effect of recombinant mAbs and represents a potential alternative "vaccine" strategy for those with compromised immune systems or in possible outbreak response scenarios.

AAV-monoclonal antibody expression protects mice from Ebola virus without impeding the endogenous antibody response to heterologous challenge.

Filoviruses cause severe hemorrhagic fever with case fatality rates as high as 90%. Filovirus-specific monoclonal antibodies (mAbs) confer protection in nonhuman primates as late as 5 days after challenge, and FDA-approved mAbs REGN-EB3 and mAb114 have demonstrated efficacy against Ebola virus (EBOV) infection in humans. Vectorized antibody expression mediated by adeno-associated virus (AAV) can generate protective and sustained concentrations of therapeutic mAbs in animal models for a variety of infectious diseases, including EBOV. Here we demonstrate that AAV6.2FF-mediated expression of murine IgG2a EBOV mAbs, 2G4 and 5D2, protects from mouse-adapted (MA)-EBOV infection with none of the surviving mice developing anti-VP40 antibodies above background. Protective serum concentrations of AAV6.2FF-2G4/AAV6.2FF-5D2 did not alter endogenous antibody responses to heterologous virus infection. AAV-mediated expression of EBOV mAbs 100 and 114, and pan-ebolavirus mAbs, FVM04, ADI-15878, and CA45, as human IgG1 antibodies conferred protection against MA-EBOV at low serum concentrations, with minimum protective serum levels as low as 2 µg/mL. Vectorized expression of murine IgG2a or human IgG1 mAbs led to sustained expression in the serum of mice for >400 days or for the lifetime of the animal, respectively. AAV6.2FF-mediated mAb expression offers an alternative to recombinant antibody administration in scenarios where long-term protection is preferable to passive immunization.

AAV-Vectored Expression of the Vascular Normalizing Agents 3TSR and Fc3TSR, and the Anti-Angiogenic Bevacizumab Extends Survival in a Murine Model of End-Stage Epithelial Ovarian Carcinoma.

Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.

Safety and Tolerability of the Adeno-Associated Virus Vector, AAV6.2FF, Expressing a Monoclonal Antibody in Murine and Ovine Animal Models.

Adeno-associated virus (AAV) vector mediated expression of therapeutic monoclonal antibodies is an alternative strategy to traditional vaccination to generate immunity in immunosuppressed or immunosenescent individuals. In this study, we vectorized a human monoclonal antibody (31C2) directed against the spike protein of SARS-CoV-2 and determined the safety profile of this AAV vector in mice and sheep as a large animal model. In both studies, plasma biochemical parameters and hematology were comparable to untreated controls. Except for mild myositis at the site of injection, none of the major organs revealed any signs of toxicity. AAV-mediated human IgG expression increased steadily throughout the 28-day study in sheep, resulting in peak concentrations of 21.4-46.7 µg/ mL, demonstrating practical scale up from rodent to large animal models. This alternative approach to immunity is worth further exploration after this demonstration of safety, tolerability, and scalability in a large animal model.

A phase II study of REOLYSIN® (pelareorep) in combination with carboplatin and paclitaxel for patients with advanced malignant melanoma.

REOLYSIN® (pelareorep) is an investigational new drug, consisting of a live, replication-competent, Reovirus Type 3 Dearing strain in a proprietary formulation. Several preclinical and clinical trials with REOLYSIN® on a wide range of cancer indications have demonstrated antineoplastic activity on cells with activated RAS-signaling pathway. Furthermore, long-term survival benefits were evident in post-treatment patients indicating a potential antitumor immune response triggered by REOLYSIN®. Numerous mono and/or combination therapy studies with the agent showed a consistent safety profile. The current study is a phase II, single-arm, open label trial of REOLYSIN® in combination with carboplatin and paclitaxel for patients with advanced melanoma. Results from the 14 patients enrolled in the study exhibited no grade 4 adverse events or deaths but manageable grade-3 toxicities commonly attributed to REOLYSIN®, including pyrexia, chills, myalgia, pain, fatigue, and nausea. The number of treatment cycles ranged from 2 to 20 with a median of 6 cycles. The study met its treatment and efficacy goal for the first stage with three partial responses (ORR was 21%). No complete responses were noted. The median PFS and OS were 5.2 and 10.9 months, respectively. The 1-year OS was 43% with a disease control rate of 85%. In conclusion, REOLYSIN® combined with carboplatin and paclitaxel is a safe and potentially efficacious therapy for patients with advanced malignant melanoma. Additional combination studies using REOLYSIN® with chemo/immunotherapy drugs may support more favorable outcomes for patients in this indication.

The Influence of a Vocalized Checklist on Detection of Multiple Abnormalities in Chest Radiography.

RATIONALE AND OBJECTIVES: Although a checklist has been recommended for preventing satisfaction of search (SOS) errors, a previous research study did not demonstrate that benefit. However, observers in that study had to turn away from the image display to use the checklist. The current study tested a vocalized checklist to avoid this constraint. MATERIALS AND METHODS: A total of 64 chest computed radiographs, half containing various "test" abnormalities, were read twice by 20 radiologists, once with and once without the addition of a simulated pulmonary nodule. Readers used a vocalized checklist-directing search. Receiver operating characteristic (ROC) detection accuracy and decision thresholds were analyzed to study the effects of adding the nodule on detecting the test abnormalities. RESULTS: Adding nodules induced a substantial reluctance to report the other abnormalities (P < 0.001), as had been the case in the most recent study of the SOS effect in radiography. CONCLUSIONS: The vocalized checklist did not reduce nor eliminate the SOS effect on readiness to report further abnormalities. Although useful for organizing search and reporting, particularly among students, a vocalized checklist does not prevent SOS effects.

Novel Logistic Regression Model of Chest CT Attenuation Coefficient Distributions for the Automated Detection of Abnormal (Emphysema or ILD) Versus Normal Lung.

RATIONALE AND OBJECTIVES: We evaluated the role of automated quantitative computed tomography (CT) scan interpretation algorithm in detecting interstitial lung disease (ILD) and/or emphysema in a sample of elderly subjects with mild lung disease. We hypothesized that the quantification and distributions of CT attenuation values on lung CT, over a subset of Hounsfield units (HUs) range (-1000 HU, 0 HU), can differentiate early or mild disease from normal lung. MATERIALS AND METHODS: We compared the results of quantitative spiral rapid end-exhalation (functional residual capacity, FRC) and end-inhalation (total lung capacity, TLC) CT scan analyses of 52 subjects with radiographic evidence of mild fibrotic lung disease to the results of 17 normal subjects. Several CT value distributions were explored, including (1) that from the peripheral lung taken at TLC (with peels at 15 or 65 mm), (2) the ratio of (1) to that from the core of lung, and (3) the ratio of (2) to its FRC counterpart. We developed a fused-lasso logistic regression model that can automatically identify sub-intervals of -1000 HU and 0 HU over which a CT value distribution provides optimal discrimination between abnormal and normal scans. RESULTS: The fused-lasso logistic regression model based on (2) with 15-mm peel identified the relative frequency of CT values of over -1000 HU and -900 and those over -450 HU and -200 HU as a means of discriminating abnormal versus normal lung, resulting in a zero out-sample false-positive rate, and 15% false-negative rate of that was lowered to 12% by pooling information. CONCLUSIONS: We demonstrated the potential usefulness of this novel quantitative imaging analysis method in discriminating ILD and/or emphysema from normal lungs.

Satisfaction of Search in Chest Radiography 2015.

RATIONALE AND OBJECTIVES: Two decades have passed since the publication of laboratory studies of satisfaction of search (SOS) in chest radiography. Those studies were performed using film. The current investigation tests for SOS effects in computed radiography of the chest. METHODS: Sixty-four chest computed radiographs half demonstrating various "test" abnormalities were read twice by 20 radiologists, once with and once without the addition of a simulated pulmonary nodule. Receiver-operating characteristic detection accuracy and decision thresholds were analyzed to study the effects of adding the nodule on detecting the test abnormalities. Results of previous studies were reanalyzed using similar modern techniques. RESULTS: In the present study, adding nodules did not influence detection accuracy for the other abnormalities (P = .93), but did induce a reluctance to report them (P < .001). Adding nodules did not affect inspection time (P = .58) so the reluctance to report was not associated with reduced search. Reanalysis revealed a similar decision threshold shift that had not been recognized in the early studies of SOS in chest radiography (P < .01) in addition to reduced detection accuracy (P < .01). CONCLUSIONS: The nature of SOS in chest radiography has changed, but it is not clear why. ADVANCES IN KNOWLEDGE: SOS may be changing as a function of changes in radiology education and practice.

The oncolytic virus, pelareorep, as a novel anticancer agent: a review.

Pelareorep (REOLYSIN®) is an investigational new drug, a proprietary formulation consisting of a live, replication-competent, naturally occurring Reovirus Type 3 Dearing strain. Through several preclinical studies it was determined that reovirus can exhibit profound cytotoxic effects on cancer cells predominantly with an activated RAS-signalling pathway. Moreover, it was discovered that reoviruses can "hitchhike" on peripheral blood mononuclear cells and dendritic cells, thereby evading neutralizing antibodies of the host immune system. Cell carriage, targeted delivery, triggering host immune response and other inherent characteristics of the reovirus led to its further advancement into cancer therapy. When injected into Sprague-Dawley rats, the viral routes of clearance, predominantly through the spleen and liver, remained consistent with earlier studies. Toxicology findings were considered incidental and not associated with pelareorep when tested in animal models. Pelareorep demonstrated a high level of homogeneity at the amino acid level and genetic stability when compared to the master and working virus banks. The drug is manufactured in a 100 L bioreactor after which it is purified and formulated for use in pre-clinical, clinical and research studies. Over the past few decades, we have witnessed a paradigm shift from conventional therapy to the conceivable use of oncolytic viruses for the treatment of cancer. This review will detail pre-clinical evidence of anticancer activity of pelareorep that has led to extensive clinical development. Several Phase I-II clinical trials have been completed or are ongoing in cancer patients on a broad spectrum of solid tumors and hematologic malignancies.

Oncolytics Biotech® Inc.: REOLYSIN® for melanoma therapy.

Oncolytics Biotech® Inc. is focused on the development of oncolytic viruses for use as cancer therapeutics. The Company's lead product, REOLYSIN®, a proprietary formulation of the human reovirus, is in late-stage clinical testing in a range of cancers. REOLYSIN has been utilized in nearly 30 clinical trials including translational, Phase I, Phase II (single arm and randomized) and Phase III studies in a broad range of cancer indications. To date, Oncolytics has been issued more than 370 patents worldwide and has more than 230 pending applications. Issued patent claims pertaining to the reovirus cover: compositions of matter comprising reovirus; pharmaceutical use of reoviruses to treat neoplasia and cellular proliferative diseases and combination therapy with radiation, chemotherapy and/or immune suppressants.

Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production.

REOLYSIN (pelareorep) is a proprietary isolate of the reovirus T3D (Type 3 Dearing) strain which is currently being tested in clinical trials as an anticancer therapeutic agent. Reovirus genomes are composed of ten segments of double-stranded ribonucleic acid (RNA) characterized by genome size: large (L1, L2, and L3), medium (M1, M2, and M3), and small (S1, S2, S3, and S4). The objective of this work was to evaluate the homogeneity and genetic stability of REOLYSIN. Sanger sequencing (SS) performed on test articles derived from the Master Virus Bank (MVB) and Working Virus Bank (WVB) identified many modifications when compared to GenBank reference sequences. Massively parallel sequencing (MPS) using Roche-454 sequencing was performed on REOLYSIN (100 L scale) and resulted in 69,821,115 bases and an average of 335 bases per read. Twenty-nine high confidence differences relative to the GenBank reference sequence were identified in REOLYSIN by MPS. Of those, 27 were previously identified by SS in the virus bank-derived test articles. Of the remaining two nucleotide differences, one was predicted to be silent at the amino acid level (L3 genome-T3163C, codon 1054, 86% of the population was "T" and 13% of the population were reported as "C"). The other modification was in the noncoding region (M1 genome-A2284A to A2284G), and A2284G was present in 97% of the population. The results obtained from MPS were comparable to those from SS; both demonstrate a high level of homogeneity at the amino acid level and genetic stability of REOLYSIN. Finally, phylogenetic analysis of the REOLYSIN L1 genome segment showed close evolutionary relationship with its human homologs, serotypes Lang and Dearing.

Bio-distribution study of Reolysin® (pelareorep) through a single intravenous infusion in Sprague-Dawley rats.

Numerous pre-clinical and clinical studies on reovirus have generated valuable information which supports the use of this orphan virus as an investigational drug for cancer treatment. Reolysin® (pelareorep) is a clinical formulation of the human Reovirus Type 3 Dearing strain. The clinical safety and efficacy of Reolysin® in humans is being tested on an assortment of cancer indications as a mono and/or combination therapy. Reovirus has many inherent characteristics that make it a potential candidate for virotherapy, including: the rapid and natural spread through the haematogenous route, the ability to overcome immunological barriers thereby reaching tumor sites, and being replication-competent. The purpose of this study was to elucidate the bio-distribution pattern of Reolysin® in healthy Sprague-Dawley rats. Following a single 15-min intravenous infusion via the tail vein in Sprague-Dawley rats, the levels of virus genome were determined in 16 organs/tissues by RT-qPCR (Reverse Transcriptase- Quantitative Polymerase Chain Reaction) over a 336 h (Day 15) incubation regime. Consistent with previous studies, maximal reovirus RNA levels were observed in the spleen; indicating its involvement in viral uptake and clearance, followed by heart, ovaries, tail (infusion site), liver and lungs. All the organs/tissues demonstrated unquantifiable levels of reovirus genome at the end of incubation, suggesting substantial to complete viral clearance. Several studies in the last decade have described the use of reovirus for treating ovarian cancers. An increase of reovirus genome in ovaries at 24 h post infection was noted. The results will aid in the design of additional exploratory clinical trials for Reolysin®.

Satisfaction of search from detection of pulmonary nodules in computed tomography of the chest.

RATIONALE AND OBJECTIVES: We tested whether satisfaction of search (SOS) effects that occur in computed tomography (CT) examination of the chest on detection of native abnormalities are produced by the addition of simulated pulmonary nodules. MATERIALS AND METHODS: Two experiments were conducted. In the first experiment, 70 CT examinations, half that demonstrated diverse, subtle abnormalities and half that demonstrated no native lesions, were read by 18 radiology residents and fellows under two experimental conditions: presented with and without pulmonary nodules. In a second experiment, many of the examinations were replaced to include more salient native abnormalities. This set was read by 14 additional radiology residents and fellows. In both experiments, detection of the natural abnormalities was studied. Receiver operating characteristic (ROC) curve areas for each reader-treatment combination were estimated using empirical and proper ROC models. Additional analyses focused on decision thresholds and visual search time on abnormality-free CT slice ranges. Institutional review board approval and informed consent from 32 participants were obtained. RESULTS: Observers more often missed diverse native abnormalities when pulmonary nodules were added, but also made fewer false-positive responses. There was no change in ROC area, but decision criteria grew more conservative. The SOS effect on decision thresholds was accompanied by a reduction in search time on abnormality-free CT slice ranges. CONCLUSION: The SOS effect in CT examination of the chest is similar to that found in contrast examination of the abdomen, involving induced visual neglect.

REO-001: A phase I trial of percutaneous intralesional administration of reovirus type 3 dearing (Reolysin®) in patients with advanced solid tumors.

PURPOSE: This open-labeled, phase I clinical trial was designed to determine the safety and tolerability of percutaneous intralesional administration of wild-type oncolytic revovirus type 3 Dearing (Reolysin®) in cancer patients with accessible and evaluable disease, who had otherwise failed to improve on standard cancer interventions. EXPERIMENTAL DESIGN: An escalating dose of Reolysin® starting from up to 10(10) plague forming units (PFU) was administered to each cohort of three patients per dose level. Viral shedding, reovirus neutralizing antibody response, toxicity and clinical response were assessed. RESULTS: Nineteen patients with various advanced solid tumors were treated. The most common toxicities related to treatment were grade 2 (or less) local erythema and transient flu like symptoms. Viral shedding was not seen in cerebral spinal fluid (CSF), urine and stool samples in all patients. Rising viral antibody titres were seen in all patients. In addition, we observed some evidence of local target tumor response activity in 7/19 patients (37 %) at the end of six or more weeks follow-up, with one patient exhibiting a complete response (CR), two a partial response (PR), and four stable disease (SD) to the local injected lesion. CONCLUSIONS: Reolysin® is well tolerated given intralesionaly, with DLT/MTD not reached at a dose of 10(10) PFU. The favorable toxicity profile, lack of viral shedding and possible therapeutic activity has made this unattenuated oncolytic reovirus an attractive cancer therapeutic agent for ongoing clinical studies, including in the setting of locally advanced accessible disease for palliation of symptoms.