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.

NKp30 ‐ A prospective target for new cancer immunotherapy strategies

“…This strategy, however, presents major drawbacks regarding the production and cost of CAR‐T cells. The work of Phillips et al (2016) provided important insights regarding the activation of NK cell response with small peptides, but more affordable and comprehensive strategies should be developed in the future as part of a new branch of cancer immunotherapy.…”

“…Treatment of NK cells with this molecule induced the release of TNF‐α, proving that NKp30 may be targeted using small peptides. This work, however, failed to achieve a complete response of NK cells, as no release of IFN‐γ was triggered by the designed peptides and no data regarding the cytolytic activity of NK cells were reported (Phillips et al, 2016).…”

“…This isoform‐prognostic correlation is particularly evident in paediatric neuroblastoma patients, where the pattern of isoform expression is highly correlated to a 10‐year event‐free survival in multiple cohorts (Longo et al, 2012; Yang et al, 2019). However, this requires further investigation, as a later clinical study addressing paediatric neuroblastoma response to imatinib treatment failed to identify any correlation between NKp30 isoforms and clinical response (Phillips, Romeo, Bitsaktsis, & Sabatino, 2016).…”

“…On the other hand, the other known tumour‐derived ligand of NKp30, B7‐H6, is able to induce cytokine release both in its soluble and membrane‐bound forms (Gutierrez‐Franco et al, 2018; Phillips et al, 2016). Membrane expression of B7‐H6 has been widely implicated in the sensitivity of cancer cells to NK cell‐mediated cytolysis (Cao et al, 2015).…”

B7-H6, an immunoligand for the natural killer cell activating receptor NKp30, reveals inhibitory effects on cell proliferation and migration, but not apoptosis, in cervical cancer derived-cell lines

“…B7 family members are cell-surface protein ligands that are expressed on antigen presenting cells as well as on tumors, which bind to their respective receptors on T lymphocytes and provide positive or negative signals to promote or down-regulate T cell responses [ 7 , 8 ]. In contrast to the well-known antigen presenting cell expressed B7.1 and B7.2, which provide the crucial second activation signal for T cells [ 9 ], B7-H6 is a distinct member of the B7 family that has been shown to be a functional ligand for the NK cell-activating receptor NKp30 that mediates NK cell-dependent killing [ 10 , 11 ]. This ligand is selectively expressed by tumor cells (such as lymphoma, melanoma, leukemia, and gastric carcinoma), but not by healthy cells, thus making it an important marker and target on tumor cells [ 12 , 13 ].…”