Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines.

Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. 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). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

Identification of a neo-epitope dominating endogenous CD8 T cell responses to MC-38 colorectal cancer.

The murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous CD8+ T cell responses to MC-38 neo-antigens in tumor-bearing mice and after anti-PD-L1 checkpoint therapy. Through combination of whole-exome sequencing analysis with mass spectrometry of MHC class I eluted peptides we could identify eight candidate epitopes. Of these, a neo-epitope encoded by a point-mutation in the sequence of the ribosomal protein L18 (Rpl18) strongly dominated the CD8+ T cell response to our MC-38 cell-line in comparison to a previously described neo-epitope in the Adpgk protein. Therapeutic vaccination with synthetic peptides induced CD8+ T cell responses against the mutated Rpl18 epitope, which controlled tumor growth in vivo. This immunologically dominant response to mutated Rpl18 is of great importance in the development and optimization of immunotherapeutic strategies with the MC-38 tumor model.