BackgroundThe generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed.MethodsA unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood.ResultsHere, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists.ConclusionsWe identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, “DualPep-Shine”, which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.