Design and development of an RNA vaccine against tumor-mediated angiogenesis and metastasis targeting a KLH-Adrenomedullin fusion protein
Adrenomedullin (AM) is an autocrine/paracrine growth factor as well as a crucial regulator of angiogenesis and immune response. AM is overexpressed by most solid tumors, which makes it a good target for anti-tumor therapy. In this study, we designed and tested a mRNA vaccine directed against a fusion antigen composed by a small piece of keyhole limpet hemocyanin (KLH), as a hapten, and mouse AM. The in vitro-synthesized mRNA was encapsulated in lipid nanoparticles (LNPs) and injected in C57BL/6J mice. Empty LNPs were used as a negative control. After five immunizations, B16- F10 melanoma cells were injected through the tail vein to induce lung metastases. In addition, transgenic mice expressing green fluorescent protein in the blood vessels (SMAA-GFP) were also immunized with both LNP types to assess the effect of the vaccine in subcutaneous tumors and potential side-effects of the vaccine on normal blood vessel formation. Antibody titers against AM and the number of CD8+ T cells were significantly higher in AMimmunized mice than in negative controls. Furthermore, the number and size of the lung metastases, as well as the number of blood vessels, were significantly reduced in the AMimmunized group. Furthermore, the volume of subcutaneous tumors was significantly reduced with significant differences observed in the number of blood vessels. In addition, no significant differences were observed in the number and distribution of blood vessels in normal tissues and organs after immunization of the SMAA-GFP animals. In summary, we have shown that a mRNA vaccine against the fusion KLH-AM peptide was able to break peripheral immunotolerance and induce a specific response against the angiogenic factor AM thus reducing tumor burden in two melanoma tumor models in the absence of disturbances to normal blood vessels. The study results indicate a great translational potential and possibility to use it in combinational anti-tumor treatment settings, as well as possibility to overcome challenges of existing therapies targeting angiogenesis and AM itself.
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