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.

中文翻译：

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豇豆花叶病毒瘤内免疫治疗长期贮存后仍能保持稳定性和疗效


豇豆花叶病毒 （CPMV） 作为瘤内免疫疗法已显示出卓越的免疫刺激和疗效，为其临床转化提供了有力的论据。任何新候选药物的一个重要考虑因素是药物及其制剂的长期稳定性。因此，我们的实验室在从超低温冰箱到加热培养箱的多种温度条件下评估了 CPMV 制剂（含和不含蔗糖防腐剂）的物理稳定性和生物活性，即抗肿瘤效力，为期 9 个月。我们发现非冷藏温度 37°C 和室温迅速导致 CPMV 不稳定，仅 1 周后蛋白质和 RNA 的显着降解就证明了这一点。在 4°C 下冷藏可延长物理稳定性，但 6 个月和 9 个月后出现颗粒破裂和 RNA 逃逸的迹象。在整个研究期间，储存在冷冻条件（包括 -20°C、-80°C 和液体 N2）中的 CPMV 保持完整，并与新鲜 CPMV 的特性相匹配。使用小鼠真皮黑色素瘤模型评估生物活性，疗效遵循观察到的物理稳定性趋势：与新鲜制备的制剂相比，储存在冷藏和温暖条件下的 CPMV 表现出较低的抗肿瘤功效。同时，冷冻储存的 CPMV 的性能与新鲜纯化的 CPMV 相似，导致肿瘤生长减少并延长生存期。因此，数据表明，在寒冷或冷冻条件下长期储存的 CPMV 保持稳定和有效，为推进这种强大的植物病毒进行翻译提供了额外的支持。