Comparative Evaluation of Plant-Derived Virus-like Particles as Intratumoral Immunotherapy Agents

Background: Plant-derived virus-like particles (VLPs) are emerging nanoplatforms for local cancer immunotherapy, yet their relative performance across structurally distinct particles remains insufficiently defined. Methods: We performed a comparative benchmarking study of eleven plant-derived VLPs spanning diverse architectures and functional properties using an integrated workflow of physicochemical characterization, immune-functional profiling, and in vivo evaluation. All VLPs were produced in endotoxin-minimized ClearColi BL21 (DE3), enabling assessment of intrinsic particle-associated immunostimulatory activity with reduced bacterial endotoxin confounding. Results: In vitro, several VLPs stimulated macrophage-associated responses and enhanced tumor cell killing, although classical M1/M2 polarization markers in RAW264.7 cells did not consistently predict functional cytotoxicity. In a subset of candidates, HEK-TLR3 reporter activity varied substantially under RNA-normalized conditions and was not predicted solely by total RNA content or apparent RNA size distribution. Five candidates were advanced to intratumoral evaluation in the male-derived B16-F10 melanoma model, where CCMV-ss and CMVtt showed trends toward reduced tumor progression and increased immune cell infiltration in male mice. Furthermore, host sex-associated differences in baseline immune features were observed, though these must be interpreted with caution given the H-Y antigen-driven immunogenicity inherent to the male-derived B16-F10 model in female hosts. Conclusions: This study establishes a standardized comparative framework linking plant VLP properties with immune-functional performance and identifies CCMV-ss and CMVtt as promising candidates for further development as locally administered cancer immunotherapy nanoplatforms.