Background/Objectives: Nucleic acid-based anticancer vaccines are becoming a very active field in the fight against cancer. Here, our goal was to generate an oral DNA vaccine targeting the angiogenic peptide, proadrenomedullin N-terminal 20 peptide (PAMP). Methods: An expression plasmid (PcPAMP) was generated by fusing the tetanus toxin epitopes P2 and P30 to the mouse PAMP sequence to counteract self-tolerance, and the empty plasmid was used as a negative control (PcNeg). The plasmids were introduced into Salmonella typhimurium bacteria that were then transformed into bacterial ghosts. C57BL/6J mice were orally immunized with the ghosts five times at 2-week intervals. Then, B16-F10 melanoma cells were injected into the tail vein to generate lung metastases. Furthermore, naïve CD4⁺ T cells were exposed to PAMP, and their secretome was analyzed by proximity extension assays. Results: Significant levels of anti-PAMP immunoglobulins were detected in the blood of PcPAMP-vaccinated mice and their levels of spleen CD8⁺ T cells were significantly higher than in those treated with PcNeg, indicating that self-tolerance was effectively broken. Although the number and size of lung metastases was similar between both experimental groups, there was a significant reduction in intratumoral angiogenesis and in cancer cell proliferation index in the PcPAMP group. Furthermore, these animals showed an intense infiltration of lymphocytes, including regulatory T cells, and M2-like macrophages into the metastases, that was not evident in the PcNeg group. In addition, PAMP induced upregulation of IL1β, IL6, IL7, IL12, IL27, TNFα, and FGF21, and downregulation of IL16 in naïve CD4⁺ T cells. Conclusions: Although the vaccine was not effective in reducing tumor growth, new proliferative and immune functions have been described for PAMP. These new functions include induction of melanoma proliferation and modulation of lymphocyte and macrophage tumor infiltration dynamics.

Adrenomedullin (AM) is a regulatory peptide that stimulates proliferation, migration, and invasion of melanoma cells, and promotes neovascularization within the tumor microenvironment, making it a compelling therapeutic target in melanoma and other cancers. As a continuation of our previous study on a metastatic tumor model, here we tested an mRNA vaccine encoding a fusion antigen comprising keyhole limpet hemocyanin (KLH) and mouse AM in a subcutaneous melanoma mouse model. In vitro synthesized mRNA was encapsulated in lipid nanoparticles (LNPs) and administered to C57BL/6J mice; empty LNPs served as negative controls. After a four-dose immunization schedule, B16-F10 melanoma cells were injected subcutaneously, followed by a fifth immunization. Mice were sacrificed once tumors reached humane endpoints. Immunization led to a significant increase in anti-AM IgG titers (p = 0.033) and CD8⁺ T cell (p = 0.049) numbers in treated mice. Tumor initiation was significantly delayed (p = 0.005) and subcutaneous tumor volume was reduced (p = 0.0004) compared to controls. A marked decrease in the area occupied by tumor blood vessels (p = 0.028) was also observed, with no signs of systemic toxicity or weight loss. In addition, there was no significant impairment of Ki67⁺ tumor cell proliferation nor changes in the tumor infiltration of CD4⁺, CD8⁺, FoxP3⁺ nor Arg1⁺ cells. The vaccine also proved highly stable at 4 °C, in the absence of cryoprotectants, for more than a month. In summary, we confirmed that a KLH-AM mRNA vaccine is very stable and can elicit humoral and cellular immune responses, inhibit angiogenesis, and delay tumor growth in subcutaneous melanoma, without inducing an immunosuppressive tumor microenvironment (TME), further supporting mRNA vaccines targeting AM as an attractive immunotherapeutic approach.