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. 2025 Oct;20(19):2425-2431.
doi: 10.1080/17435889.2025.2544522. Epub 2025 Aug 9.

Storage stability of cowpea mosaic virus cancer drug candidate - a two-year update

Andrea Simms  1   2   3   4 , Narek Minasov  1   2   3   4 , Nicole F Steinmetz  1   2   3   4   5   6   7   8
Affiliations

Storage stability of cowpea mosaic virus cancer drug candidate - a two-year update

Andrea Simms et al. Nanomedicine (Lond). 2025 Oct.

Abstract

Aim: To evaluate the structural stability and biological activity of cowpea mosaic virus (CPMV) after 2 years of storage.

Materials and methods: CPMV was stored at room temperature (RT), 4°C, and -20°C for 2 years. Structural stability was assayed using dynamic light scattering (DLS), size exclusion chromatography (SEC), native and denaturing gel electrophoresis, and transmission electron microscopy (TEM). Biological activity was assessed by anti-tumor efficacy studies using a B16F10 dermal mouse melanoma model and by a cowpea plant infection challenge.

Results: Storage at -20°C preserved CPMV's structural properties and biological activity. Storage at 4°C caused aggregation, S-protein cleavage, and RNA degradation - samples stored at 4°C had decreased anti-tumor efficacy yet retained plant infectivity. CPMV stored at RT was degraded and lost biological activity.

Conclusion: Frozen storage allows CPMV to maintain structural and biological stability for at least 2 years.

Keywords: Cowpea mosaic virus; cancer immunotherapy; intratumoral immunotherapy; storage stability; translational drug development.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Dr Steinmetz is a co-founder of, has equity in, and has a financial interest in PlantiosX Inc. Dr Steinmetz is a co-founder of, has equity in, and has a financial interest in Mosaic ImmunoEngineering Inc. Dr Steinmetz is a co-founder and manager of Pokometz Scientific LLC, under which she is a paid consultant to Flagship Labs 95 Inc.

The other authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Figures

Figure 1.
Figure 1.
Characterization of fresh CPMV or CPMV after 2 years of storage at room temperature (RT), 4°C, or − 20°C. CPMV was analyzed by dynamic light scattering (DLS) (n = 3 runs) (A) Size exclusion chromatography (SEC) (B) Transmission electron microscopy (TEM) (C) SDS-PAGE (D) or native agarose gel electrophoresis (E) In DLS and SEC graphs, values marked in green indicate particles within acceptable ranges and red indicates values out of acceptable range.
Figure 2.
Figure 2.
Biological activity of CPMV after 2 years of storage. (A) CPMV (fresh, RT, 4°C, or − 20°C) was mechanically inoculated in black-eyed pea leaves and infection was allowed to proceed for 14 days (n = 31–40 individually infected leaves per group). Infection is indicated by a mosaic of yellow spots. (B–C) female C57BL6 mice were inoculated intradermally with 200,000 B16F10 cells on day 0; then, mice were treated intratumorally starting on day 8 with 100 µg of fresh, RT, 4°C, or − 20°C CPMV (n = 10 per group). Mice received treatments weekly for a total of three treatments. Panel (B) shows overall tumor growth (left) and survival (right), with growth curves ending when there are fewer than three mice alive in any given group. Panel (C) shows individual mouse tumor growth. Mice were euthanized when tumors reached 1000 mm3. Statistical significance of tumor size was calculated via two-way ANOVA with Tukey’s multiple comparisons test and a single pooled variance, and survival comparisons were calculated with Mantel-Cox log rank test; ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤ 0.0001. Statistical analyses were carried out using GraphPad prism software version 10.4.1.
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