461. The Testing Strategy to Determine the Pharmacological Activity of Gene Therapy Drug Product (rAd-IFN) to Treat Intravesical Non-Muscle Invasive Bladder Cancer

Abstract

rAd-IFN is a recombinant adenoviral gene therapy vector encoding IFNα2b gene for the treatment of refractory non-muscle invasive bladder cancer. The vector transduces bladder wall cells where IFNα2b gene is expressed leading to death of cancer cells. The advanced testing strategy to determine the pharmacological activity of rAd-IFN drug product involves three key assays: 1. Infectious titer of the virus, quantitative assay 2. Expression of the transgene (IFNα2b), semiquantitative assay 3. Potency (IFNα2b mediated cell killing), quantitative assay The infectivity and transgene expression assays have been performed for batch release and stability monitoring of activity during Phase 2 and will remain unchanged in principle for Phase 3 and commercial use. In the infectivity assay the cells supporting adenovirus replication are infected with three concentrations of adenovirus and left to produce the virus for two days. The percentage of infected cells is then determined with a flow cytometer utilizing a fluorescently conjugated antibody against an adenoviral structural protein. Samples are analysed in parallel with a reference standard and infectivity is given as relative Infectious Units / ml. In expression assay, the IFNα expression capability of the virus preparation is determined by infecting IFN insensitive cells with the rAd-IFN virus and the concentration of produced IFNα is measured with a commercial IFNα ELISA (enzyme-linked immunosorbent assay) from cell culture supernatants For Phase 3 a new potency assay is developed and added to release and stability testing in order to provide evidence that batches of rAd-IFN are able to produce active IFNα2b which has a relevant pharmacological effect. In this assay cells are transduced using multiple dilutions of reference standard and test samples leading to expression of IFNα2b and subsequent cell death. Cell killing efficiency is determined using colorimetric method measuring dehydrogenase activity of the living cells. Relative potency of test sample is determined against reference standard response curve after testing parallelism by equivalence test. All activity assays will be fully validated according to ICH Q2 (R1) prior to release testing of Phase 3 clinical study material (Accuracy, Precision, Specificity, Linearity and Range, System Suitability and Robustness). The three validated assays will provide enhanced quantitative measure of biologic function of the rAd-IFN vector and thus demonstrate the quality and efficacy of drug product batches.