(M1130-09-56) Development of an Industry Ready Cell-Based Potency Method for an Oncolytic Virus Immunotherapy

Purpose: Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn’t represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput.

Methods: To address the above-mentioned unmet needs, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. Master and working stocks of SK-MEL-28 cells, a melanoma cell line established from patient-derived tumor samples (ATCC HTB-72, SK1980-524), were prepared in-house, and cultured in Eagles Essential Media (EMEM) + 10% FBS and 1% Penicillin-Streptomycin. Test articles (TA) of CVA21 virus included in-house generated Drug Substances (DS), Drug Product (DP), Working and Master Virus Seeds used at various stages of method development.

Results: In this study, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/ week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method.

Conclusion: In summary, a potency assay was developed for CVA21, and the steps used to optimize and validate the assay method were described, as well as the statistical methods for data analysis. Although tight clinical program timelines may lead to elimination or compaction of certain method development steps, when completed, these steps greatly reduce the risk of assay performance problems in the long run. This step-by-step guide can serve as a strategy for others considering assay method development for infectious viral products.