Cowpea mosaic virus intratumoral immunotherapy maintains stability and efficacy after long-term storage

Cowpea mosaic virus (CPMV) has demonstrated superior immune stimulation and efficacy as an intratumoral immunotherapy, providing a strong argument for its clinical translation. One important consideration for any new drug candidate is the long-term stability of the drug and its formulation. Therefore, our lab has evaluated the physical stability and biological activity, that is, anti-tumor potency, of formulations of CPMV in buffer (with and without a sucrose preservative) in multiple temperature conditions ranging from ultralow freezers to a heated incubator over a period of 9 months. We found that non-refrigerated temperatures 37°C and room temperature quickly led to CPMV destabilization, as evidenced by significant protein and RNA degradation after just 1 week. Refrigerated storage at 4°C extended physical stability, though signs of particle breakage and RNA escape appeared after 6 and 9 months. CPMV stored in frozen conditions, including −20°C, −80°C, and liquid N₂, remained intact and matched the characteristics of fresh CPMV throughout the duration of the study. The biological activity was evaluated using a murine dermal melanoma model, and efficacy followed the observed trends in physical stability: CPMV stored in refrigerated and warmer conditions exhibited decreased anti-tumor efficacy compared to freshly prepared formulations. Meanwhile, frozen-stored CPMV performed similarly to freshly purified CPMV, resulting in reduced tumor growth and extended survival. Data, therefore, indicates that CPMV stored long-term in cold or frozen conditions remains stable and efficacious, providing additional support to advance this powerful plant virus to translation.