Stability and safety key factors of the oncolytic protoparvovirus H-1 from manufacturing to human application.

The oncolytic rodent protoparvovirus H-1PV has been successfully used in phase I/II clinical trials to treat recurrent glioblastoma multiforme and pancreatic cancer. The present work focuses on the stability and environmental safety of the H-1PV drug product from production up to its use in patients. We identified hold-steps in manufacturing for up to 3 months and showed 7-years stability for the optimal product formulation. Stress testing via UV, temperature, and pH also determined that the drug product is stable. De- and rehydration for lyophilization simulation are possible without infectious virus loss. Furthermore, we prove in-use stability for 4 days at room temperature and show no virus adsorption to injection devices, guaranteeing the correct administration dose. Iodixanol in the formulation, resulting in high viscosity, protects H-1PV against UV and some disinfectants. Nonetheless, H-1PV is depleted with rapid heat deactivation, autoclavation, and nanofiltration. Assessment of chemical disinfectants that are currently recommended by the Robert Koch-Institute demonstrated that ethanol-based hand disinfectants are not effective; however, aldehyde-based disinfectants for surfaces and instruments demonstrate sufficient H-1PV deactivation in aqueous formulations by 4 to 6 log10. With these results, we could establish a specific hygiene plan for all involved facilities from manufacturing to patient application. Overall, using 48% Iodixanol in Visipaque/Ringer as a drug formulation stabilizes H-1PV infectivity over years and protects against virus loss from short-term UV, low pH, and temperature exposure. KEY POINTS: • Optimal formulation of drug product protects the H-1PV protoparvovirus against UV, temperatures up to 50 °C, and low pH (> 1.25), stabilizing the virus during manufacturing, storage, transport, and application. • H-1PV is stable during in-use and does not adsorb to injection devices during patient administration. • Hygiene plan for H-1PV with physicochemical methods has been established.

Phase 2 Trial of Oncolytic H-1 Parvovirus Therapy Shows Safety and Signs of Immune System Activation in Patients With Metastatic Pancreatic Ductal Adenocarcinoma.

PURPOSE: To investigate the safety, clinical efficacy, virus pharmacokinetics, shedding, and immune response after administration of an oncolytic parvovirus (H-1PV, ParvOryx) to patients with metastatic pancreatic ductal adenocarcinoma (PDAC) refractory to first-line therapy. PATIENTS AND METHODS: This is a noncontrolled, single-arm, open-label, dose-escalating, single-center clinical trial. Seven patients with PDAC and at least one liver metastasis were included. ParvOryx was administered intravenously on 4 consecutive days and as an intralesional injection, 6 to 13 days thereafter. Altogether, three escalating dose levels were investigated. In addition, gemcitabine treatment was initiated on day 28. RESULTS: ParvOryx showed excellent tolerability with no dose-limiting toxicities. One patient had a confirmed partial response and one patient revealed an unconfirmed partial response according to RECIST criteria. Both patients showed remarkably long surivial of 326 and 555 days, respectively. Investigation of pharmacokinetics and virus shedding revealed dose dependency with no excretion of active virus particles in saliva or urine and very limited excretion in feces. H-1PV nucleic acids were detected in tumor samples of four patients. All patients showed T-cell responses to viral proteins. An interesting immunologic pattern developed in tumor tissues and in blood of both patients with partial response suggesting immune activation after administration of ParvOryx. CONCLUSIONS: The trial met all primary objectives, revealed no environmental risks, and indicated favorable immune modulation after administration of ParvOryx. It can be considered a good basis for further systematic clinical development alone or in combination with immunomodulatory compounds.

Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial.

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.

A non-controlled, single arm, open label, phase II study of intravenous and intratumoral administration of ParvOryx in patients with metastatic, inoperable pancreatic cancer: ParvOryx02 protocol.

BACKGROUND: Metastatic pancreatic cancer has a dismal prognosis, with a mean six-month progression-free survival of approximately 50% and a median survival of about 11 months. Despite intensive research, only slight improvements of clinical outcome could be achieved over the last decades. Hence, new and innovative therapeutic strategies are urgently required. ParvOryx is a drug product containing native parvovirus H-1 (H-1PV). Since H-1PV was shown to exert pronounced anti-neoplastic effects in pre-clinical models of pancreatic cancer, the drug appears to be a promising candidate for treatment of this malignancy. METHODS: ParvOryx02 is a non-controlled, single arm, open label, dose-escalating, single center trial. In total seven patients with pancreatic cancer showing at least one hepatic metastasis are to be treated with escalating doses of ParvOryx according to the following schedule: i) 40% of the total dose infused intravenously in equal fractions on four consecutive days, ii) 60% of the total dose injected on a single occasion directly into the hepatic metastasis at varying intervals after intravenous infusions. The main eligibility criteria are: age ≥ 18 years, disease progression despite first-line chemotherapy, and at least one hepatic metastasis. Since it is the second trial within the drug development program, the study primarily explores safety and tolerability after further dose escalation of ParvOryx. The secondary objectives are related to the evaluation of certain aspects of anti-tumor activity and clinical efficacy of the drug. DISCUSSION: This trial strongly contributes to the clinical development program of ParvOryx. The individual hazards for patients included in the current study and the environmental risks are addressed and counteracted adequately. Besides information on safety and tolerability of the treatment after further dose escalation, thorough evaluations of pharmacokinetics and intratumoral spread as well as proof-of-concept (PoC) in pancreatic cancer will be gained in the course of the trial. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT02653313 , Registration date: Dec. 4th, 2015.

Bioavailability, biodistribution, and CNS toxicity of clinical-grade parvovirus H1 after intravenous and intracerebral injection in rats.

The autonomous parvovirus H1 (H1PV) is transmitted in rodent populations. The natural host is the rat, in which H1PV infection is pathogenic only in fetuses and newborns. H1PV infection of human cancer cells leads to strong oncolytic effects in preclinical models. In preparation for a clinical trial of H1PV injection in patients with malignant brain tumors, H1PV had to be prepared to Good Manufacturing Practice standards, including extensive toxicology testing in rats. Because the trial involves direct intracerebral injection of H1PV into the tumor and around the resection cavity, possible toxicity to CNS tissue had to be investigated. In addition, quantitative blood levels and the tissue distribution of H1PV after single intracerebral or intravenous injection were measured. Direct injection of H1PV into rat brain at 3 dose levels (maximum, 7.96 × 107 pfu) did not cause any macroscopic or histologic pathology. Furthermore, H1PV infection of the brain did not alter central or autonomous nervous system function. H1PV DNA was detected in almost all organs at 6 h, 48 h, and 14 d after intravenous and intracerebral injection, with the highest levels in liver and spleen. H1PV concentrations in most organs were similar after intravenous and intracerebral injection, indicating high permeability of the blood-brain barrier for this small virus. The current results demonstrate wide organ distribution of H1PV after intravenous or intracerebral injection, confirm that H1PV is nonpathogenic in adult rats even after direct injection into the brain, and form the basis for the ongoing ParvOryx01 clinical trial.

Pathology, organ distribution, and immune response after single and repeated intravenous injection of rats with clinical-grade parvovirus H1.

Parvovirus H1 (H1PV) is an autonomous parvovirus that is transmitted in rodent populations. Its natural host is rats. H1PV infection is nonpathogenic except in rat and hamster fetuses and newborns. H1PV infection of human cancer cells caused strong oncolytic effects in preclinical models. For a clinical trial of H1PV in patients with brain tumors, clinical-grade H1PV was produced according to Good Manufacturing Practices. This report focuses on results obtained after a single high-dose intravenous injection of highly purified H1PV in 30 rats and multiple (n = 17) intravenous injections at 3 dose levels in 223 rats. In both studies, no virus-related mortality or macroscopic organ changes related to H1PV occurred. Histopathology after multiple virus injections revealed minimal diffuse bile duct hyperplasia in livers of animals of the highest dose group and germinal center development in spleens of animals from the high-dose group. Liver changes were reversible within a 2-wk recovery period after the last injection. Hematology, blood chemistry, and coagulation analyses did not reveal significant toxicologic changes due to H1PV. Virus injection stimulated the production of IgG antibodies but did not alter mononuclear cell function or induce cytokine release. PCR analysis showed dose-dependent levels of viral genomes in all organs tested. The virus was excreted primarily through feces. These data provide important information regarding H1PV infection in its natural host. Due to the confirmation of the favorable safety profile of H1PV in a permissive animal model, a phase I/IIa clinical trial of H1PV in brain tumor patients could be initiated.

Phase I/IIa study of intratumoral/intracerebral or intravenous/intracerebral administration of Parvovirus H-1 (ParvOryx) in patients with progressive primary or recurrent glioblastoma multiforme: ParvOryx01 protocol.

BACKGROUND: The treatment of patients with malignant brain tumors remains a major oncological problem. The median survival of patients with glioblastoma multiforme (GBM), the most malignant type, is only 15 months after initial diagnosis and even less after tumor recurrence. Improvements of standard treatment including surgery and radio-chemotherapy have not lead to major improvements. Therefore, alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are under investigation. Preclinical data of oncolytic parvovirus H-1 (H-1PV) infection of glioma cells demonstrated strong cytotoxic and oncosuppressing effects, leading to a phase I/IIa trial of H-1PV in patients with recurrent GBM (ParvOryx01). ParvOryx01 is the first trial with a replication competent oncolytic virus in Germany. METHODS: ParvOryx01 is an open, non-controlled, two groups, intra-group dose escalation, single center, phase I/IIa trial. 18 patients with recurrent GBM will be treated in 2 groups of 9 patients each. Treatment group 1 will first receive H-1PV by intratumoral injection and second by administration into the walls of the tumor cavity during tumor resection. In treatment group 2 the virus will initially be injected intravenously and afterwards, identical to group 1, into the surrounding brain tissue during tumor removal. Main eligibility criteria are: age of 18 years, unifocal recurrent GBM, amenable to complete or subtotal resection. Dose escalation will be based on the Continual Reassessment Method. The primary objective of the trial is local and systemic safety and tolerability and to determine the maximum tolerated dose (MTD). Secondary objectives are proof of concept (PoC) and Progression-free Survival (PFS) up to 6 months. DISCUSSION: This is the first trial with H-1PV in patients with recurrent GBM. The risks for the participants appear well predictable and justified. Furthermore, ParvOryx01 will be the first assessment of combined intratumoral and intravenous application of an oncolytic virus. Due to its study design the trial will not only generate data on the local effect of H-1PV but it will also investigate the penetration of H-1PV into the tumor after systemic delivery and obtain safety data from systemic delivery possibly supporting clinical trials with H-1PV in other, non-CNS malignancies. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01301430.

Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud.

A unique limb phenotype is described in a radiation-induced mutant mouse resulting from an inversion of a proximal segment of chromosome 5. The limb phenotype in the homozygous mutant presents with two anterior skeletal elements in the zeugopod but no posterior bone, hence the name replicated anterior zeugopod, raz. The zeugopod phenotype is accompanied by symmetrical central polydactyly of hand and foot. The chromosomal inversion includes the Shh gene and the regulatory locus, located approximately 1 Mb away, within the Lmbr1 gene. In homozygous mutants, the expression of Shh mRNA and Shh protein is severely downregulated to about 20% of wild-type limb buds, but Shh expression appears normal throughout the remainder of the embryo. Correspondingly, Gli3 expression is upregulated and posteriorly expanded in the raz/raz limb bud. We propose that the double anterior zeugopod and symmetrical central polydactyly are due to an increased and uniform concentration of the Gli3 repressor form because of lowered Shh signaling.

Developmental regulation of hyaluronan-binding protein (RHAMM/IHABP) expression in early bovine embryos.

Hyaluronan or hyaluronic acid (HA) is a normal component of mammalian follicular, oviduct, and uterine fluids. Granulosa and expanding cumulus cells secrete large amounts of HA, and when HA is added in maturation and culture media, it improves the developmental potential of oocytes and embryos. HA regulates gene expression, signaling, proliferation, motility, adhesion, and morphogenesis. Many of these biological activities of HA are mediated through binding to the receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP). We evaluated the presence and dynamics of RHAMM/IHABP mRNA and protein expression in different stages of in vitro-produced bovine embryos using quantitative reverse transcriptase-real time-polymerase chain reaction and immunohistochemistry. We also analyzed the effects of different culture systems on the relative abundance of RHAMM/IHABP transcripts. RHAMM/IHABP mRNA levels decreased from the 2-cell to the 16-cell stage, increased again at the morula stage, and reached their highest level at the expanded blastocyst stage. RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower in embryos recovered in serum-containing medium than in embryos from serum-free media. Immunohistochemistry revealed the presence of RHAMM/IHABP first in 8-cell stages. Whereas RHAMM staining in 8-cell and morula stages was intense, it was weaker in blastocysts. Embryonic secretion of HA increased from the 2-cell stage until the 8-cell stage and then decreased in 16-cell embryos. After this, HA secretion increased in expanded and hatched blastocyst stages. These data suggest that the positive effects of HA on in vitro-produced bovine embryos may be mediated at least in part by RHAMM/IHABP.

Diethylnitrosamine induces long-lasting re-expression of insulin-like growth factor II during early stages of liver carcinogenesis in mice.

The expression of the insulin-like growth factor II (IGF-II) gene (Igf2) in rodents is completely abrogated in almost all adult tissues. A prominent exception are neoplasms in which IGF-II frequently serves as an autocrine growth factor. We have investigated the potential role of Igf2 expression during liver carcinogenesis. After application of diethylnitrosamine (DEN) preneoplastic foci and adenomas emerged in liver tissue of wild-type and phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II transgenic mice. Surprisingly, number and size of preneoplastic foci were not significantly increased in PEPCK-IGF-II mice as compared with wild-type animals. In situ preparation showed that early adenomas expressed Igf2 transcripts. Reverse transcriptase polymerase chain reaction (RT-PCR) and restriction enzyme analysis confirmed that DEN treatment had indeed reactivated the hepatic expression of murine Igf2 in control mice in a dose-dependent manner. This re-expression of Igf2 persisted for at least 18 months. Species-specific RT-PCR analyses also revealed the presence of murine Igf2 mRNAs in some PEPCK-IGF-II mice. A similar reactivation of Igf2 was detected in bovine growth hormone transgenic mice which develop hepatocellular neoplasms with high frequency. Our results suggest that reactivation of Igf2 is an early event during hepatocarcinogenesis in mice. Its appearance in two independent animal models suggests that Igf2 may be important at pivotal checkpoints of hepatocarcinogenesis.