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1.
Appl Microbiol Biotechnol ; 107(15): 4777-4787, 2023 Aug.
Article in English | MEDLINE | ID: mdl-37209160

ABSTRACT

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.


Subject(s)
Glioblastoma , H-1 parvovirus , Oncolytic Virotherapy , Oncolytic Viruses , Pancreatic Neoplasms , Humans , Oncolytic Viruses/physiology , Oncolytic Virotherapy/methods , H-1 parvovirus/physiology , Pancreatic Neoplasms/therapy
2.
Appl Microbiol Biotechnol ; 105(24): 9113-9124, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34782922

ABSTRACT

The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. KEY POINTS: • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.


Subject(s)
H-1 parvovirus , Oncolytic Viruses , Bioreactors , Cell Culture Techniques , Culture Media , Oncolytic Viruses/genetics
3.
Sci Transl Med ; 13(609): eabe6805, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34516824

ABSTRACT

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body's vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor­ß (TGFß) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)­dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis.


Subject(s)
Glycoproteins , Neoplasms , Glycoproteins/genetics , Humans , Neoplasms/genetics
4.
Clin Cancer Res ; 27(20): 5546-5556, 2021 10 15.
Article in English | MEDLINE | ID: mdl-34426438

ABSTRACT

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.


Subject(s)
Adenocarcinoma/secondary , Adenocarcinoma/therapy , Carcinoma, Pancreatic Ductal/secondary , Carcinoma, Pancreatic Ductal/therapy , H-1 parvovirus , Immune System/immunology , Oncolytic Virotherapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/therapy , Aged , Humans , Middle Aged , Oncolytic Virotherapy/adverse effects
5.
Viruses ; 13(6)2021 05 28.
Article in English | MEDLINE | ID: mdl-34071585

ABSTRACT

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.


Subject(s)
H-1 parvovirus/immunology , H-1 parvovirus/pathogenicity , Homeostasis/immunology , Immunity, Innate , Interferons/immunology , Pancreatic Neoplasms/virology , Cell Death/immunology , Cell Line, Tumor , Cytokines , Humans , Leukocytes, Mononuclear/virology , Oncolytic Viruses/genetics , Oncolytic Viruses/immunology , Oncolytic Viruses/pathogenicity , Parvoviridae Infections/complications , Parvoviridae Infections/virology
6.
J Virol ; 94(19)2020 09 15.
Article in English | MEDLINE | ID: mdl-32669341

ABSTRACT

Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.


Subject(s)
Capsid/metabolism , Dependovirus/genetics , Dependovirus/physiology , Sumoylation/physiology , Transduction, Genetic , A549 Cells , Capsid Proteins/genetics , Capsid Proteins/metabolism , Genetic Therapy , Genetic Vectors/genetics , HEK293 Cells , HeLa Cells , Humans , Sumoylation/genetics
7.
Methods Mol Biol ; 2058: 295-306, 2020.
Article in English | MEDLINE | ID: mdl-31486047

ABSTRACT

Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection-for the first time in humans-of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.


Subject(s)
Brain Neoplasms/diagnosis , DNA, Viral , Genetic Vectors , H-1 parvovirus , In Situ Hybridization, Fluorescence , Oncolytic Viruses , Brain Neoplasms/therapy , Fluorescent Antibody Technique , Genetic Vectors/genetics , H-1 parvovirus/genetics , H-1 parvovirus/immunology , Humans , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , Oncolytic Viruses/immunology , Tumor Microenvironment , Virus Replication
8.
Neuromuscul Disord ; 29(3): 231-241, 2019 03.
Article in English | MEDLINE | ID: mdl-30782477

ABSTRACT

So far effective strategies to treat cardiomyopathy in patients with muscular dystrophies are still not clearly defined. Previously, treatment with ß-blockers showed beneficial effects on the development of cardiomyopathy in dystrophin-deficient (mdx) mice, but not in δ-sarcoglycan-deficient (Sgcd-/-) mice. We therefore aimed to study a more specific approach to target maladaptive ß-adrenergic signalling in these mice. It has been shown that lowering cardiac G-protein-coupled-receptor-kinase-2 (GRK2) activity with ßARKct expression, a peptide inhibitor of protein-coupled-receptor-kinase-2 (GRK2), results in improvement of heart failure in several different animal models. We therefore investigated whether adeno-associated virus type 9 (AAV9)-mediated gene delivery of ßARKct, could ameliorate cardiac pathology in mdx and Sgcd-/- mice. We found that long-term treatment with AAV9- ßARKct-cDNA with a cardiac-specific promoter significantly improves left ventricular systolic function and reduces myocardial hypertrophy in mdx mice, whereas only mild beneficial effects on cardiac function is observed in Sgcd-/- mice. Interestingly, in contrast to mdx mice neither GRK2 nor nuclear-factor-kappaB (NFκB) were upregulated in Sgcd-/- mice. Taken together, effectiveness of AAV-mediated ßARKct therapy may vary between different genetic mutations and presumably depend on the state of adrenergic dysregulation mediated through the upregulation of GRK2.


Subject(s)
Cardiomyopathies/genetics , Dependovirus , Dystrophin/deficiency , Muscular Dystrophies/genetics , Sarcoglycans/genetics , Animals , Dependovirus/genetics , Dystrophin/genetics , Genetic Therapy/methods , Heart/physiopathology , Heart Failure/genetics , Heart Failure/physiopathology , Mice, Inbred mdx , Mice, Transgenic , Muscular Dystrophies/metabolism , Ventricular Function, Left/genetics
9.
Viruses ; 10(6)2018 06 03.
Article in English | MEDLINE | ID: mdl-29865280

ABSTRACT

About 70% of all Ewing sarcoma (EWS) patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV) to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU)/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.


Subject(s)
Apoptosis , H-1 parvovirus/physiology , Oncolytic Virotherapy , Sarcoma, Ewing/therapy , Sarcoma, Ewing/virology , Animals , Cell Cycle , Cell Line, Tumor , Cell Survival , Female , Humans , Mice, Nude , Oncolytic Viruses/physiology , Parvovirus , Virus Replication , Xenograft Model Antitumor Assays
10.
Mol Ther ; 25(12): 2620-2634, 2017 Dec 06.
Article in English | MEDLINE | ID: mdl-28967558

ABSTRACT

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.


Subject(s)
Genetic Therapy , Genetic Vectors/genetics , Glioblastoma/genetics , Glioblastoma/therapy , H-1 parvovirus/genetics , Oncolytic Virotherapy , Oncolytic Viruses/genetics , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Female , Gene Expression , Genetic Therapy/adverse effects , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/immunology , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Male , Middle Aged , Molecular Targeted Therapy , Oncolytic Virotherapy/adverse effects , Oncolytic Virotherapy/methods , Radiotherapy , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Transgenes , Treatment Outcome
11.
Viruses ; 9(10)2017 10 17.
Article in English | MEDLINE | ID: mdl-29039746

ABSTRACT

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.


Subject(s)
Cell Death , H-1 parvovirus/physiology , Oncolytic Viruses/physiology , Osteosarcoma/pathology , Osteosarcoma/virology , Cell Cycle , Cell Line , Cell Line, Tumor , Humans , Oncolytic Virotherapy , Virus Replication
12.
Hum Gene Ther Methods ; 28(5): 235-246, 2017 10.
Article in English | MEDLINE | ID: mdl-29048971

ABSTRACT

Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.


Subject(s)
Dependovirus/genetics , Plasmids/metabolism , Cell Culture Techniques , Centrifugation/standards , Electrophoresis, Polyacrylamide Gel , HEK293 Cells , Humans , Plasmids/analysis , Plasmids/standards , Quality Control , Real-Time Polymerase Chain Reaction , Transfection
13.
Cell Rep ; 20(6): 1422-1434, 2017 08 08.
Article in English | MEDLINE | ID: mdl-28793265

ABSTRACT

Increased pro-inflammatory signaling is a hallmark of metabolic dysfunction in obesity and diabetes. Although both inflammatory and energy substrate handling processes represent critical layers of metabolic control, their molecular integration sites remain largely unknown. Here, we identify the heterodimerization interface between the α and ß subunits of transcription factor GA-binding protein (GAbp) as a negative target of tumor necrosis factor alpha (TNF-α) signaling. TNF-α prevented GAbpα and ß complex formation via reactive oxygen species (ROS), leading to the non-energy-dependent transcriptional inactivation of AMP-activated kinase (AMPK) ß1, which was identified as a direct hepatic GAbp target. Impairment of AMPKß1, in turn, elevated downstream cellular cholesterol biosynthesis, and hepatocyte-specific ablation of GAbpα induced systemic hypercholesterolemia and early macro-vascular lesion formation in mice. As GAbpα and AMPKß1 levels were also found to correlate in obese human patients, the ROS-GAbp-AMPK pathway may represent a key component of a hepato-vascular axis in diabetic long-term complications.


Subject(s)
Atherosclerosis/metabolism , GA-Binding Protein Transcription Factor/metabolism , Hepatocytes/metabolism , Hypercholesterolemia/metabolism , Protein Kinases/metabolism , Signal Transduction , AMP-Activated Protein Kinase Kinases , Animals , Atherosclerosis/etiology , Atherosclerosis/pathology , Cell Line , Cells, Cultured , Cholesterol/metabolism , GA-Binding Protein Transcription Factor/chemistry , Hypercholesterolemia/complications , Male , Mice , Mice, Inbred C57BL , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolism
14.
BMC Cancer ; 17(1): 576, 2017 Aug 29.
Article in English | MEDLINE | ID: mdl-28851316

ABSTRACT

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.


Subject(s)
H-1 parvovirus/physiology , Oncolytic Virotherapy/methods , Pancreatic Neoplasms/drug therapy , Administration, Intravenous , Dose-Response Relationship, Drug , Female , Humans , Injections, Intralesional , Male , Neoplasm Metastasis , Oncolytic Virotherapy/adverse effects , Oncolytic Viruses/physiology , Sample Size , Survival Analysis , Treatment Outcome
15.
Appl Microbiol Biotechnol ; 101(8): 3143-3152, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28091791

ABSTRACT

The rodent protoparvovirus H-1PV, with its oncolytic and oncosuppressive properties, is a promising anticancer agent currently under testing in clinical trials. This explains the current demand for a scalable, good manufacturing practice-compatible virus purification process yielding high-grade pure infectious particles and overcoming the limitations of the current system based on density gradient centrifugation. We describe here a scalable process offering high purity and recovery. Taking advantage of the isoelectric point difference between full and empty particles, it eliminates most empty particles. Full particles have a significantly higher cationic charge than empty ones, with an isoelectric point of 5.8-6.2 versus 6.3 (as determined by isoelectric focusing and chromatofocusing). Thanks to this difference, infectious full particles can be separated from empty particles and most protein impurities by Convective interaction media® diethylaminoethyl (DEAE) anion exchange chromatography: applying unpurified H-1PV to the column in 0.15 M NaCl leaves, the former on the column and the latter in the flow through. The full particles are then recovered by elution with 0.25 M NaCl. The whole large-scale purification process involves filtration, single-step DEAE anion exchange chromatography, buffer exchange by cross-flow filtration, and final formulation in Visipaque/Ringer solution. It results in 98% contaminating protein removal and 96% empty particle elimination. The final infectious particle concentration reaches 3.5E10 plaque forming units (PFU)/ml, with a specific activity of 6.8E11 PFU/mg protein. Overall recovery is over 40%. The newly established method is suitable for use in commercial production.


Subject(s)
Capsid/chemistry , Chromatography, Ion Exchange/methods , H-1 parvovirus/chemistry , H-1 parvovirus/isolation & purification , Isoelectric Focusing/methods , Animals , Capsid/virology , Cations , Filtration/methods , H-1 parvovirus/ultrastructure , Isoelectric Point , Microscopy, Electron , Rats
16.
Viruses ; 8(5)2016 05 19.
Article in English | MEDLINE | ID: mdl-27213425

ABSTRACT

Combining virus-induced cytotoxic and immunotherapeutic effects, oncolytic virotherapy represents a promising therapeutic approach for high-grade glioma (HGG). A clinical trial has recently provided evidence for the clinical safety of the oncolytic parvovirus H-1 (H-1PV) in adult glioblastoma relapse patients. The present study assesses the efficacy of H-1PV in eliminating HGG initiating cells. H-1PV was able to enter and to transduce all HGG neurosphere culture models (n = 6), including cultures derived from adult glioblastoma, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Cytotoxic effects induced by the virus have been observed in all HGG neurospheres at half maximal inhibitory concentration (IC50) doses of input virus between 1 and 10 plaque forming units per cell. H-1PV infection at this dose range was able to prevent tumorigenicity of NCH421k glioblastoma multiforme (GBM) "stem-like" cells in NOD/SCID mice. Interestingly NCH421R, an isogenic subclone with equal capacity of xenograft formation, but resistant to H-1PV infection could be isolated from the parental NCH421k culture. To reveal changes in gene expression associated with H-1PV resistance we performed a comparative gene expression analysis in these subclones. Several dysregulated genes encoding receptor proteins, endocytosis factors or regulators innate antiviral responses were identified and represent intriguing candidates for to further study molecular mechanisms of H-1PV resistance.


Subject(s)
H-1 parvovirus/growth & development , Neuroglia/physiology , Neuroglia/virology , Oncolytic Viruses/growth & development , Stem Cells/physiology , Stem Cells/virology , Animals , Disease Models, Animal , Gene Expression Profiling , Glioma/therapy , Heterografts , Humans , Mice, Inbred NOD , Mice, SCID , Models, Theoretical , Oncolytic Virotherapy/methods
17.
Mol Ther Methods Clin Dev ; 3: 16018, 2016.
Article in English | MEDLINE | ID: mdl-27088104

ABSTRACT

Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention.

18.
J Virol Methods ; 229: 48-59, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26658622

ABSTRACT

The promising anticancer properties of rodent protoparvoviruses, notably H-1PV, have led to their clinical testing. This makes it necessary to produce highly pure, well-characterized virus batches in sufficient quantity. The present work focused on developing standardized production, purification, and characterization procedures as a basis for exploiting H-1PV both preclinically and in clinical trials for anticancer virotherapy. Two infection and two virus purification strategies were tested and the resulting virus preparations compared for their purity and full-, infectious-, and empty-particle contents. The adopted production process, which involves culturing and infecting NB-324K cells in 10-layer CellSTACK(®) chambers (1×10(3) infectious units per infected cell), is simple, scalable, and reproducible. Downstream processing to eliminate contaminating DNA and protein includes DNAse treatment, filtration, and two Iodixanol density-gradient centrifugations, the first gradient being a step gradient and the second, either a step (1×10(10) PFU/ml) or a continuous gradient (3×10(11) PFU/ml). A procedure was also developed for obtaining infectious particle-free preparations of empty virions for research purposes: cesium chloride density gradient centrifugation followed by UV irradiation (1×10(14) physical particles/ml). For quick, sensitive determination of physical particles (and hence, particle-to-infectivity ratios), a "Capsid-ELISA" was developed, based on a novel monoclonal antibody that specifically targets assembled capsids.


Subject(s)
Centrifugation, Density Gradient/methods , Filtration/methods , Parvovirinae/growth & development , Parvovirinae/isolation & purification , Virus Cultivation/methods , Cell Line , Centrifugation, Density Gradient/standards , Disinfection/methods , Epithelial Cells/virology , Filtration/standards , Humans , Viral Load/methods , Virus Cultivation/standards
19.
Comp Med ; 65(1): 36-45, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25730755

ABSTRACT

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.


Subject(s)
Central Nervous System/virology , H-1 parvovirus/pathogenicity , Oncolytic Virotherapy/methods , Animals , Biological Availability , Central Nervous System/pathology , DNA, Viral/metabolism , Drug Evaluation, Preclinical , Injections, Intravenous , Liver/virology , Oncolytic Virotherapy/standards , Rats , Spleen/virology , Time Factors , Viral Load
20.
Comp Med ; 65(1): 23-35, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25730754

ABSTRACT

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.


Subject(s)
Genome, Viral/genetics , H-1 parvovirus/pathogenicity , Oncolytic Virotherapy/methods , Parvoviridae Infections/immunology , Parvoviridae Infections/pathology , Animals , Blood Chemical Analysis , Blood Coagulation Tests , Dose-Response Relationship, Immunologic , Drug Evaluation, Preclinical , Feces/virology , H-1 parvovirus/genetics , Immunoglobulin G/immunology , Injections, Intravenous , Liver/pathology , Polymerase Chain Reaction , Rats , Spleen/pathology
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