Multiplexed Targeting of Barrett's Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft in Vivo and Human Specimens ex Vivo

Abstract

Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease that is rising rapidly in incidence and has poor prognosis. We developed a heterobivalent peptide to target detection of early Barrett's neoplasia by combining monomer heptapeptides specific for either EGFR or ErbB2 in a heterodimer configuration. The structure of a triethylene glycol linker was optimized to maximize binding interactions to the surface receptors on cells. The Cy5.5-labeled heterodimer QRH*-KSP*-E3-Cy5.5 demonstrated specific binding to each target and showed 3-fold greater fluorescence intensity and 2-fold higher affinity compared with those of either monomer alone. Peak uptake in xenograft tumors was observed at 2 h postinjection with systemic clearance by ∼24 h in vivo. Furthermore, ligand binding was evaluated on human esophageal specimens ex vivo, and 88% sensitivity and 87% specificity were found for the detection of either high-grade dysplasia (HGD) or EAC. This peptide heterodimer shows promise for targeted detection of early Barrett's neoplasia in clinical study.

Figure 1.. Design of peptide heterodimer.

Chemical structures for heptapeptide monomers QRHKPRE (QRH*) and KSPNPRF (KSP*) are shown along with arrangement of heterodimer configuration with variable length linkers.

Figure 2.. Optimization of peptide heterodimer.

On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A) E2, B) Hex, C) E3, D) E6, and E) E10 to the surface (arrow) of SKBr3 cells can be seen. F) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P-values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.

Figure 3.. Characterization of peptide heterodimer.

In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P=3.6×10⁻⁴ and P=7.8×10⁻³, respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be k_d = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min⁻¹ (4.5 min) versus 0.21 min⁻¹ (4.8 min) and 0.35 min⁻¹ (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.

Figure 4.. No effect of peptide heterodimer on cell signaling.

We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.

Figure 5.. Pharmacokinetics of peptide heterodimer.

A) Representative whole-body NIR fluorescence images show uptake of targeted peptide heterodimer QRH*-KSP*-E3-Cy5.5 in xenograft tumor (arrow) over time. B) (GGGAGGG)₂KK-Cy5.5 was used as control. C) Quantitative analysis shows peak mean signal from (n = 6) tumors at 2 hours following iv injection. A mean (±SD) T/B ratio of 2.39±0.52 and 1.43±0.28 was measured at the tumor for the targeted (n = 6) and control (n = 6) peptides, respectively. Signal returns to baseline by ~24 hours. D) The mean fluorescence intensity from individual tumors at 2 hours is significantly greater for the targeted versus control peptide, P=2.9×10⁻³ by unpaired t-test. E) Unlabeled QRH*, KSP*, and both (QRH*, KSP*) monomer peptides were injected iv prior to heterodimer for blocking. F) Quantified results show significant reduction in mean intensity from tumor (n = 3).

Figure 6.. Biodistribution of peptide heterodimer.

Representative fluorescence images are shown from excised mouse organs at ~2 hours post-injection of A) targeted peptide heterodimer QRH*-KSP*-E3-Cy5.5 and B) control peptide (GGGAGGG)₂KK-Cy5.5. C) Fluorescent signals were quantified from individual organs (n = 6) for each peptide. The mean fluorescence intensity for targeted was significantly greater than that for control, 5.28±1.14 versus 2.24±0.82, P=5.5×10⁻⁴ by unpaired t-test.

Figure 7.. Specific binding of peptide heterodimer to Barrett’s neoplasia.

On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.

Figure 8.. Co-localization of peptide heterodimer and antibody binding to Barrett’s neoplasia.

A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm² placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P-value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.