B7H6-derived peptides trigger TNF-α-dependent immunostimulatory activity of lymphocytic NK92-MI cells

Abstract The rise of biologics that can stimulate immune responses towards the eradication of tumors has led to the evolution of cancer‐based immunotherapy. Representatively, B7H6 has been recently identified as a protein ligand on tumor cells that binds specifically to the NKp30 receptor and triggers NK cell‐derived cytokine production, which ultimately leads to tumor cell lysis and death. In an effort to develop effective immunotherapy approaches, the rational design of a novel class of immunostimulatory peptides (IPs) derived from the binding interface of B7H6:NKp30 is described in this study. The IPs comprised the B7H6 active site sequence for NKp30 binding and immunostimulatory activity. An aminohexanoic acid linker was also introduced at the N ‐terminus of the peptides for FITC‐labeling by Fmoc‐solid phase peptide synthesis. The peptides were characterized by LCMS to confirm identities and purities >95%. The secondary structures of the peptides were examined by CD spectroscopy in H 2 O, PBS and a H 2 O:TFE mixture which demonstrated versatile peptide structures which transitioned from random coil (H 2 O) to α ‐helical (PBS) and turn‐type (H 2 O:TFE) conformations. Their biological properties were then evaluated by flow cytometry, enzyme‐linked immunosorbent assays (ELISAs), and cell death assays. The occupancy of the synthetic peptides to a human NK cell line demonstrated comparable binding relative to the natural NKp30 ligand, B7H6, and the human anti‐NKp30 monoclonal antibody (mAb), in a concentration dependent manner. A competitive binding assay between the human anti‐NKp30 mAb or B7H6, and the synthetic peptides, demonstrated partial displacement of the ligands upon anti‐NKp30 mAb treatment, suggesting NKp30 receptor specificities by the synthetic peptides. Moreover, the immunostimulatory activity of B7H6 was demonstrated by the secretion of the pro‐inflammatory cytokines tumor necrosis factor‐alfa (TNF‐α) and interferon gamma (IFN‐γ) by the human NK cell line. The immunostimulatory effects of IPs on the NK cells was assessed by the production of TNF‐α alone as IFN‐γ was undetectable. In a cell death assay, the IPs were found to be nontoxic, without any observable evidence of early or late stage apoptosis within the NK92‐MI cells. Taking these findings together, this novel class of synthetic peptides may prove to be a promising lead in the development of a peptide‐based immunotherapy approach, especially against B7H6 expressing tumors. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 658–672, 2016.