title: 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 H2 O, PBS and a H2 O:TFE mixture which demonstrated versatile peptide structures which transitioned from random coil (H2 O) to α-helical (PBS) and turn-type (H2 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.
authors.affiliation: Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ, 07079.
authors.forename: Mariana
authors.initials: M
authors.lastname: Phillips
authors.affiliation: Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ, 07079.
authors.forename: Francesca
authors.initials: F
authors.lastname: Romeo
authors.affiliation: Department of Biological Sciences, Seton Hall University, South Orange, NJ, 07079. constantine.bitsaktsis@shu.edu.
authors.forename: Constantine
authors.initials: C
authors.lastname: Bitsaktsis
authors.affiliation: Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ, 07079. david.sabatino@shu.edu.
authors.forename: David
authors.initials: D
authors.lastname: Sabatino
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000494:pharmacology*; D060887:B7 Antigens / Q000737:chemistry*; D002460:Cell Line; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D055628:Natural Cytotoxicity Triggering Receptor 3 / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D060887:B7 Antigens; C573415:IFNG protein, human; C431710:NCR3 protein, human; C542363:NCR3LG1 protein, human; D055628:Natural Cytotoxicity Triggering Receptor 3; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: B7H6; NK92MI cells; NKp30; cancer-targeting immunostimulatory peptides; solid-phase peptide synthesis