Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment

Abstract

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

FIG. 1.

Chromatogram of the recombinant vesicular stomatitis virus (rVSV) pilot lot. The culture supernatant was loaded onto the column and eluted with 1.2 M NaCl. Color images available online at www.liebertonline.com/hum.

FIG. 2.

Schematic representation of the expansion of the 293 cell train used for the 50-liter rVSV GMP production lot. Cells from the 293 master cell bank were thawed and expanded for the manufacture of VSV. Cells were passaged twice per week with trypsin. An additional 10-layer Cell Factory (CF) was seeded for each week's infection in order to be able to determine the number of cells in each CF (not indicated). Additional flasks (2 × T-175) were also seeded as part of the parallel culture for each week's manufacturing production (not indicated). Color images available online at www.liebertonline.com/hum.

FIG. 3.

Representative chromatogram of the rVSV GMP lot production. Each subbatch of culture supernatant was loaded onto the column and eluted with 1.2 M NaCl. The chromatogram shown is from one of the five subbatches and is representative of all five. Color images available online at www.liebertonline.com/hum.

FIG. 4.

A sodium dodecyl sulfate–polyacrylamide gel of the rVSV(MD51)-M3 clinical lot. Major bands corresponding to the five VSV proteins (L, G, N, P, and M) were present at the expected sizes.

FIG. 5.

Electron micrograph of rVSV particles. An aliquot of the clinical lot was submitted to a contract facility for examination and quantification of viral particles by transmission electron microscopy. A representative image shows the unique bullet shape associated with VSV. The arrowhead indicates a sample rod-shaped structure. Scale bar: 100 nm.