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.