Self-assembling freeze-dried vaccine for short HLA-A2-restricted melanoma peptide epitopes

Peptide-based cancer vaccines are a promising immunotherapy modality. In this study, a facile lyophilization method was developed to formulate tumor-associated peptide antigens into multiplex vaccines. Lipids, antigens, and adjuvants are dissolved in a lyophilization solvent, sterile filtered, and freeze-dried, with the resulting lyophilized cake able to be reconstituted within seconds to form a self-assembled liposome-displayed peptide vaccine. The addition of 5% mannitol maintains the structural integrity of the freeze-dried cake, while 2% polysorbate 80 facilitates self-assembly of ∼300 nm liposomes and reduces the hemolysis caused by the included QS21 adjuvant. The formulations include cobalt porphyrin–phospholipid (CoPoP) to couple soluble peptides to liposomes, improving the immunogenicity of the peptide epitopes compared to formulations lacking CoPoP or to admixtures with polyinosinic:polycytidylic acid (pIC). A transgenic chimeric MHC mouse model was used to assess the immunogenicity of lyophilized peptide formulations with melanoma-associated antigens previously used in human testing. The lyophilized vaccine enhanced the immunogenicity of an included Melan-A₂₇ epitope compared to a non-lyophilized formulation. Even after expanding the number of included peptides to 13, the freeze-dried formulation maintained a consistent particle size and immune response. In the B16F10 mouse model, the freeze-dried vaccine induced similar tumor inhibition to its liquid counterpart. These results establish a facile lyophilization methodology to advance short peptide antigens with applications in multiplexed cancer vaccines.