In Situ Vaccination with a Vpr-Derived Peptide Elicits Systemic Antitumor Immunity by Improving Tumor Immunogenicity

Background: Cancer vaccines represent a groundbreaking advancement in cancer immunotherapy, utilizing tumor antigens to induce tumor-specific immune responses. However, challenges like tumor-induced immune resistance and technical barriers limit the widespread application of predefined antigen vaccines. Here, we investigated the potential of viral protein R (Vpr) peptides as effective candidates for constructing anonymous antigen vaccines in situ by directly injecting at the tumor site and releasing whole-tumor antigens, inducing robust anti-tumor immune responses to overcome the limitations of predefined antigen vaccines. Methods: The cytotoxic effects of Vpr peptides were evaluated using the CCK8 reagent kit. Membrane penetration ability of Vpr peptides was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. EGFR levels in the cell culture supernatants of cells treated with Vpr peptides were evaluated using an ELISA. Surface exposure of CRT on the tumor cell surface was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. The secretion levels of ATP from tumor cells were evaluated using an ATP assay kit. HMGB1 release was evaluated using an ELISA. Mouse (Male C57BL/6 mice aged 4 weeks) MC38 and LLC bilateral subcutaneous tumor models were established to evaluate the therapeutic effects of Vpr peptides through in situ vaccination. Proteomic analysis was performed to explore the mechanism of anti-tumor activity of Vpr peptides. Results: Four Vpr peptides were designed and synthesized, with P1 and P4 exhibiting cytotoxic effects on tumor cells, inducing apoptosis and immunogenic cell death. In mouse tumor models, in situ vaccination with Vpr peptide significantly inhibited tumor growth and activated various immune cells. High-dose P1 monotherapy demonstrated potent anti-tumor effects, activating DCs, T cells, and macrophages. Combining ISV of P1 with a CD47 inhibitor SIRPαFc fusion protein showed potent distant tumor suppression effects. Proteomic analysis suggested that Vpr peptides exerted anti-tumor effects by disrupting tumor cell morphology, movement, and adhesion, and promoting immune cell infiltration. Conclusions: The designed Vpr peptides show promise as candidates for in situ vaccination, with significant anti-tumor effects, immune activation, and favorable safety profiles observed in mouse models. In situ vaccination with Vpr-derived peptides represents a potential approach for cancer immunotherapy.