Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides

Abstract

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78⁺ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78⁺ /B7H6^- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

Keywords: cancer immunotherapy; cancer-targeting peptides; immunostimulatory peptides; peptide vaccines; synthetic antibody mimics.

Figure 1.

Targeting and activating functions of CTIPs.

Figure 2.

Binding studies on: A) A549 cells treated directly with trimeric peptide 13 or anti-GRP78 Ab (baseline response, black circle) and preincubation with a GRP78 blocking peptide antagonist followed by treatment with trimeric peptide 13 or anti-GRP78 Ab (antagonist response, red square). B) NK92-MI cells treated directly with trimeric peptide 13 (peptide binding, black circle), trimeric peptide 13 treatment followed by incubation with the anti-NKp30 monoclonal antibody (peptide binding, red square and antibody binding, green triangle). Control conditions include measurement of A549 and NK92-MI cells without treatment.

Figure 3.

Visualization of tumor association of the effector NK92-MI cells towards the target A549 cells by fluorescence microscopy. A) GFP⁺ A549 tumor cells (green) B) NK92-MI cells counter-stained with the Hoechst dye (blue) in co-culture with the A549 cells without activating ligands. A549-NK92-MI co-culture with activating ligands, C) B7H6; and D) trimeric peptide 13.

Figure 4.

Time dependent (1–24 h) secretion of pro-inflammatory cytokines/chemokines (IFNγ, TNFα, and IL-8) from the effector NK92-MI cells onto the targeted A549 tumor cells with and without the activating ligands, B7H6 and trimeric peptide 13 according to the Luminex assay. Quantitative values are represented by the mean of 3 trials.

Figure 5.

A549 tumor growth in vivo: A) tumor size measurements; and B) tumor growth rate curves over a 23-day period. NSG mice were challenged with A549 tumor cells and monitored after receiving no treatment (A549), NK92-MI cells alone (A549 + NK CELLS), or peptide 13 pre-activated NK92-MI cells (PEPTIDE). ANOVA statistical analysis was performed, and the PEPTIDE group was statistically (p<0.05) and consistently lower in tumor size at all time points compared to the A549 control group. The PEPTIDE group also trended consistently lower in tumor size than the A549 + NK group but without statistical significance (p=0.6746).

Scheme 1.

Fmoc-SPPS strategy for the generation of bifunctional, Y-shape trimeric peptide 13.