Simplified monopalmitoyl Toll-like receptor 2 ligand mini-UPam for self-adjuvanting neoantigen-based synthetic cancer vaccines.

Synthetic vaccines from antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for cancer immunotherapy. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). The SPs were covalently attached to UPam, a derivative of the classic TLR2 ligand Pam3CysSK4. A disadvantage of the triply palmitoylated UPam is its high lipophilicity that precludes a universal adoption of this adjuvant for covalent modification of variable antigenic peptides rendering the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, containing only one instead of three palmitoyl chains with less impact on the solubility and other physicochemical properties of the conjugate. In this study, we used SPs containing the clinically relevant neoepitopes identified in a melanoma patient who obtained a complete response after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by solid-phase synthesis using mini-UPam building block prepared in solution and a standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated using the cancer patient's T-cells.