Multimeric Branched and Hyperbranched Peptide Ligands of the Natural Cytotoxicity Receptor NKp30 on Natural Killer Cells

Synthetic multimeric peptide ligands of the natural cytotoxicity receptor, NKp30, on natural killer cells were developed in this study. A divergent solid phase peptide synthesis strategy was optimized for the conjugation of multiple peptide ligands, based on the parent TVPLN and related permutation sequences, into branched and hyperbranched peptides for structure-activity relationship studies. According to CD spectroscopy in aqueous trifluoroethanol, the parent TVPLN peptide transitioned from a β-turn (monomer and dimer) to an extended β-sheet and a helix-type conformation in its branched (trimer) and hyperbranched (tetramer) structures. The multimeric peptides were predicted to expand the binding interface to NKp30 according to molecular modeling and docking predictions. Flow cytometry revealed greater binding activity of the trimer and tetramer ligands onto NKp30-coated beads and the natural killer cells, while being displaced by the native B7H6 ligand in competitive binding studies on the NKp30-coated beads and reducing anti-NKp30 binding on the natural killer cells, suggesting some receptor-specific binding activity. Immunostimulatory activity assays showed little secretion of TNF-α and IFN-γ from the natural killer cells following peptide treatment, with the TVPLN monomer and dimer remaining superior to the rest. Select changes in sequence compositions and the ligated multimeric peptide display had an inhibitory effect on natural killer cell activation. This result was likely due to changes in bound vs unbound multimeric peptide structures that deviated from the bioactive β-turn of the TVPLN monomer and dimer for direct peptide engagement at the NKp30 active site. Nonetheless, the novel multimeric peptides showed improved cell binding activity relative to their linear counterparts and were nontoxic at lower (10 μM) doses, making them safe and effective for structure-activity studies for the discovery of novel peptide ligands of natural killer cells.