RESUMEN
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.
Asunto(s)
Terapia Genética , Vectores Genéticos/genética , Glioblastoma/genética , Glioblastoma/terapia , Parvovirus H-1/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Expresión Génica , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Viroterapia Oncolítica/efectos adversos , Viroterapia Oncolítica/métodos , Radioterapia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Transgenes , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Centrifugación por Gradiente de Densidad/métodos , Filtración/métodos , Parvovirinae/crecimiento & desarrollo , Parvovirinae/aislamiento & purificación , Cultivo de Virus/métodos , Línea Celular , Centrifugación por Gradiente de Densidad/normas , Desinfección/métodos , Células Epiteliales/virología , Filtración/normas , Humanos , Carga Viral/métodos , Cultivo de Virus/normasRESUMEN
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.