The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents.

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.

No abstract

Cancer-based immunotherapy aims to develop biologics that can stimulate immune responses towards tumor eradication, as is the case of monoclonal antibodies (mAb) and their related analogs. Although the latter have been the most prevalently used biologics in cancer immunotherapy strategies, novel therapeutics may overcome their production, administration, and pharmacological limitations. The synthesis of intermediate size molecules, particularly chemically synthesized protein-peptide bioconjugates (PPB), possessing the targeting and effector functions of antibodies, represent a novel class of immunotherapeutics that may circumvent these limitations. In this study we proposed and executed the rational design of a novel class of cancer-targeting immunostimulatory protein-peptide conjugates (CTIPPC) that may potentially work as a synthetic antibody mimic. In this proof-of-concept, the CTIPPC aimed to target the tumor expressed GRP78, while recruiting and activating NK cells for tumor targeted immunotherapy. The sustained capabilities of the CTIPP conjugates to elicit NK-dependent immunostimmulatory activities were assessed via measuring cytokine secretion, NK cell migration, and the overall anti-tumor cytotoxic activity of NK cells. These studies shed important insights into the effective design, preparation and biological evaluation of a new class of semi-synthetic protein-peptide conjugates.

IntroductionNatural Killer (NK) cells are a class of cytotoxic lymphocytes with the ability to rapidly eliminate transformed or infected cells upon activation [1]. A key mediator of the NK cell line activity, NKp30, is a member of the natural cytotoxicity receptors (NCRs) that signal pro-inflammatory cytokine and chemokine production, as well as the release of cytotoxic agents that leads to tumor cell death [2]. The active search of NKp30-dependent immunostimulatory ligands led to chemical cross-linking studies followed by tryptic digestion, tandem mass spectrometry and proteomic analyses of the leukemia cell line K562 with soluble NKp30-Fc fusion protein which revealed B7H6 as a cell membrane expressed protein ligand of the NKp30 receptor [3]. Mechanistic studies demonstrated that upon binding of B7H6 to NKp30, the association of a transmembrane arginine residue of NKp30 to an ITAM bearing protein, such as CD3ζ, initiated a signaling cascade that resulted in the reorganization of the NK cells' cytoskeleton and initiation of Ca 2+ flux that ultimately led to the secretion of inflammatory cytokines [4]. Consequently, the discovery of the B7H6-NKp30 binding interaction offers a new opportunity in the development of tumor immunotherapy applications [5]. This study will highlight our most recent achievements in validating B7H6 as a lead protein biologic in cancer immunotherapy. Results and DiscussionFlow cytometry was used to investigate the binding interactions of the soluble free form of B7H6 with the NK92-MI cells known to overexpress the cell surface NKp30 receptor. In a direct binding assay, Alexa Fluor 488-labeled B7H6 demonstrated similar binding to NK92-MI cells' (85%) compared to the human APC-labeled antiNKp30 monoclonal antibody (95%) (Figure 1). These initial results confirmed the NK cells' binding affinity of the B7H6 ligand. A competitive binding assay was next conducted in order to determine the NKp30 receptor binding specificities of the B7H6 ligand. In this assay, the NK92-MI cells were incubated with the Alexa Fluor 488-labeled B7H6 ligand, followed by treatment with the APC-labeled antiNKp30 monoclonal antibody. Flow cytometry analyses demonstrated partial displacement of B7H6 (~70%) upon addition of the antiNKp30 monoclonal antibody (Figure 2). Interestingly, when the NK92-MI cells were first incubated with the APC-labeled antiNKp30 monoclonal antibody, followed by treatment with the Alexa Fluor 488-labeled B7H6 ligand, no displacement was observed, suggesting allosteric binding of both ligands to the NKp30 receptor. Nevertheless, the ability for B7H6 to induce inflammatory responses on the NK92-MI cells was next evaluated by ELISA. In this assay, the NK92-MI cells were left untreated (negative control), and incubated with the soluble free form of B7H6 (test sample). Following a 24 h incubation period, the cells were harvested and media was tested for the secretion of the pro-inflammatory cytokines TNF and IFNIn this assay, B7H6 was found to activate NK92-MI cells resulting in the release of ...