In situ vaccine application of inactivated CPMV nanoparticles for cancer immunotherapy.

Abstract

Cowpea mosaic virus (CPMV) is currently in the development pipeline for multiple biomedical applications, including cancer immunotherapy. In particular the application of CPMV as in situ vaccine has shown promise; here the plant viral nanoparticle is used as an adjuvant and is injected directly into a tumor to reverse immunosuppression and prime systemic anti-tumor immunity. Efficacy of this CPMV-based cancer immunotherapy has been demonstrated in multiple tumor mouse models and canine cancer patients. However, while CPMV is non-infectious to mammals, it is infectious to legumes and therefore, from a safety perspective, it is desired to develop non-infectious CPMV formulations. Non-infectious virus-like particles of CPMV devoid of nucleic acids have been produced; nevertheless, efficacy of such empty CPMV nanoparticles does not match efficacy of nucleic acid-laden CPMV. The multivalent capsid activates the innate immune system through pathogen pattern recognition receptors (PRRs) such as toll-like receptors (TLRs); the RNA cargo provides additional signaling through TLR-7/8, which boosts the efficacy of this adjuvant. Therefore, in this study, we set out to develop RNA-laden, but non-infectious CPMV. We report inactivation of CPMV using UV light and chemical inactivation using β-propiolactone (βPL) or formalin. 7.5 J cm−2 UV, 50 mM βPL or 1 mM formalin was determined to be sufficient to inactivate CPMV and prevented plant infection. We compared the immunogenicity of native CPMV and inactivated CPMV formulations in vitro and in vivo using RAW-Blue™ reporter cells and a murine syngeneic, orthotropic melanoma model (using B16F10 cells and C57BL6 mice). While the in vitro assay indicated activation of the RAW-Blue™ reporter cells by formaldehyde and UV-inactivated CPMV at levels comparable to native CPMV; βPL-inactivated CPMV appeared to have diminished activity. Tumor mouse model experiments indicate potent efficacy of the chemically-inactivated CPMV (UV-treated CPMV was not tested) leading to tumor regression and increased survival; efficacy was somewhat reduced when compared to CPMV, however these samples outperformed the empty CPMV nanoparticles. These results will facilitate the translational development of safe and potent CPMV-based cancer immunotherapies.