Treatment of neoplasms with viruses
A virus, RNA virus technology, applied in the field of using viruses to treat neoplasms, can solve the problem that no work has been performed to determine the direct tumor cytotoxic activity
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Embodiment 15
[0155] a) plaque purification to prepare a cloned virus,
[0156] b) inoculation of eggs with cloned virus,
[0157] c) Egg incubation
[0158] d) Chilled eggs
[0159] e) harvesting the allantoic fluid from the eggs and diluting the allantoic fluid with buffer,
[0160] f) Removal of cellular debris from allantoic fluid by TFF.
[0161] g) purifying the virus by TFF, and
[0162] e) Purification of virus by gel permeation chromatography.
[0163] Alternatively, the virus obtained from the gel permeation step can be concentrated with TFF.
[0164] Another embodiment of the invention relates to a method of purifying replicatively competent cloned virus from eggs or cell culture comprising the steps of purifying the virus by sequential tangential flow filtration (TFF), optionally followed by gel permeation The virus is purified by chromatography, optionally followed by concentration of the virus with TFF.
[0165] Clone virus:
[0166] A cloned virus [including paramyxov...
Embodiment 1
[0232] PPMK107 (triple plaque-purified isolate of NDV strain MK107) has selective cytotoxic activity against a variety of human cancer cells relative to normal human cells
[0233] Human tumor cells and normal cells were grown to approximately 80% confluence in 24-well tissue culture dishes. The growth medium was removed and 10-fold serial dilutions of PPMK107 were added, ranging from 10 6 Plaque forming units (PFU) / well to 10 -1 PFU / well. Control wells to which no virus was added were included in each dish. Viruses were imbibed at 37°C for 90 minutes on a rotating platform. At the end of the incubation period, the virus dilution was removed and replaced with 1 ml of growth medium. Then place the dish at 37 °C in 5% CO 2Incubate for 5 days, and then quantitatively assess the amount of cytopathic effect (CPE0). Cytotoxicity was determined by using Tetrazole) colorimetric method (Cell Titer 96, catalog#G4000, Progema Corporation, MadisonWI 53711) is measured, MTT colorimet...
Embodiment 2
[0242] Use of PPMK107 for Intratumoral Therapy of Human Tumor Xenografts (5mm) in Athymic Mice
[0243] Athymic mice were injected intradermally with 10 million human tumor cells. A single dose of PPMK107 (3x10 8 PFU) or saline. Almost all tumor types showed complete or 50% to 100% partial regression rates in mice treated with PPMK107 (see Table 7). An exception was the experiment with U87MG (Experiment I): while only one of the nine tumors treated with PPMK107 completely regressed, the other two virus-treated tumors showed 32% and 20% showed slower growth than all 8 tumors treated with saline control. There was essentially no tumor regression in the saline control treated tumors: in all these experiments (A to I listed in Table 7), only one of the 73 control tumors showed regression. These results suggest that different tumor types show responses to intratumoral treatment of PPMK107.
[0244] To detect viral replication in tumors, immunohistochemical staining for viral a...
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