Efficacy of PVX and PEGylated PVX as intratumoral immunotherapy

Abstract

Intratumoral immunotherapy harnesses the tumor microenvironment to enhance local immune activation and systemic antitumor responses. Plant virus nanoparticles have emerged as potent immunostimulatory agents for this strategy. Here, we investigate the efficacy of PEGylated potato virus X (PVX-PEG) in a B-cell lymphoma model. We synthesized PVX-PEG using bis-PEG _n -NHS esters and confirmed successful conjugation through SDS-PAGE, dynamic light scattering, and transmission electron microscopy. PEGylation improved formulation stability, as evidenced by increased thermal resistance and reduced aggregation in biological conditions. In vivo, PVX-PEG exhibited prolonged tumor retention and maintained its immunotherapeutic efficacy, comparable to native PVX. Furthermore, antibody recognition of PVX-PEG was significantly reduced, highlighting its potential for clinical translation. These results suggest that PVX-PEG retains the immunostimulatory benefits of PVX while overcoming key formulation and immunogenicity challenges, supporting its advancement as a novel intratumoral immunotherapy for lymphoma.

Fig. 1. Synthesis and characterization of PVX–PEG_n. (A) Schematic representation of the structure of PVX (left) and 13 coat protein (CP) units highlighting solvent exposed lysine residues (Lys 61, Lys 70, and Lys 177) labelled in red; the primary amine groups are labelled in green; the structure was analyzed using UCSF ChimeraX software and Protein Data Bank entry 6R7G (right). (B) SDS–PAGE gel and (C) DLS analyzing PVX and crosslinked PVX–PEG_n (n = 10, 18, 25) at different molar ratio of CP and PEG_n. The conjugation rate was calculated with Fiji ImageJ band analysis tool. (D) TEM of negatively stained PVX (left) and crosslinked PVX–PEG_n (right). (E) Turbidimetric assay (absorbance at 600 nm) to determine aggregation rate of PVX and crosslinked PVX–PEG_n heated from 25 to 80 °C for 15 min. (F) DLS of PVX and crosslinked PVX–PEG_n heated at 60 °C for 15 min.

Fig. 2. Anti-tumor efficacy of PVX and PVX–PEG₁₀ against A20 lymphoma using BALB/C mice. (A) Treatment schedule. (B) Tumor growth curve and (C) survival data were plotted for each group (**p < 0.01, ***p < 0.001). (D) Confocal microscopy of tumor sections collected 24 h post first treatment dose; stained for DAPI and PVX.

Fig. 3. PEGylation reduced antibody recognition of PVX–PEG. (A) SDS–PAGE of PVX and Cy5-labeled PVX and crosslinked PVX–PEG₁₀ imaged after Coomassie staining and under white light (left) and using a red fluorescence channel confirming conjugation of the Cy5 dye. (B) Antibody recognition sandwich assay (in triplicate) to detect binding of cy5-labeled PVX or crosslinked PVX–PEG_n to an α-PVX coated plate. Imaging data in (B) and quantitative data in (C).