Development of an oral DNA anti-cancer vaccine targeting an angiogenic peptide, proadrenomedullin N-terminal 20 peptide (PAMP) stars

Abstract

Nucleic acid-based anticancer vaccines are becoming a very active field in the fight against cancer. The goal of this thesis 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 converted 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 either into the tail vein to generate lung metastases or subcutaneously to generate cutaneous melanoma. Furthermore, naive CD4+ T cells were exposed to PAMP, and their secretome was analyzed by proximity extension assays. Results: In the lung metastasis model, a humoral and cellular immune response was evidenced by the presence of significantly higher amounts of anti-PAMP IgGs and spleen CD8+ T cells in the PcPAMP-vaccinated mice. Although the number and size of lung metastases were similar between both experimental groups, there was a notable reduction in intratumoral angiogenesis and cancer cell proliferation in the PcPAMP group. Furthermore, the treatment led to intense infiltration of lymphocytes, including regulatory T cells, and M2-like macrophages into the metastases. In the cutaneous melanoma model, similar results were obtained. Despite of a high level of anti-PAMP IgGs and a marked reduction of intratumoral angiogenesis in the PcPAMP-vaccinated mice, the treatment did not reduce tumor volume or improve survival rates. A remarkable infiltration of lymphocytes and macrophages was also observed in the tumors of vaccinated animals. Additionally, the incubation of naive CD4+ T cells with PAMP induced the up-regulation of IL-1β, IL-6, IL-7, IL-12, IL-27, TNFα, and FGF21, and the down-regulation of IL-16. 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.