Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer

Abstract

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [⁸⁹Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [⁸⁹Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Figure 1:

Discovery of potential anti-MET VHHs from a camelid antibody phage display library. (A) Schematic of an IgG antibody and VHH-Fc with their average molecular weights. (B) Screening of VHH clones for MET by ELISA. 384 unpurified VHH clones in culture supernatant were added to wells coated with MET protein. VHH binding was detected using a peroxidase conjugated anti-HA-tag monoclonal antibody and Turbo TMB reagent. 12 clones with high 420 nm Absorbance values (greater than 0.9) were identified. (C) Dilution ELISA of VHH clones demonstrated saturable binding to MET. 12 unpurified VHH were serially diluted from 1 to 0.05 relative concentration and added to human MET protein coated plates. Two clones showed saturating ELISA signals.

Figure 2:

Characterization and selection of 1E7 among three anti-VHHs with high affinity for MET. (A) VHHs specificity for human (blue), rhesus macaque (orange), and murine (purple) MET was determined by ELISA. Unpurified VHH clones were serially diluted from 1 to 0.00025 and incubated on plates that were coated with the target protein. 1E7 showed saturating ELISA signals on human MET and decreased binding to rhesus macaque and murine MET. (B) Biolayer interferometry (BLI) was used to calculate and compare 1E7, 1A2, and 2G9 binding affinities to human MET. 1E7 demonstrated the highest binding affinity, whereas 1A2 and 2G9 showed weaker binding to human MET.

Figure 3:

In vitro analysis of 1E7-Fc binding specifically to MET-positive cell lines. (A) 1E7-Fc selectivity for MET-expressing cells was assessed by flow cytometry. Cells were stained with (green) or without (grey) 1E7-Fc at 1 ug/mL. Positive binding is seen in EBC-1, UW-Lung-21, and HeLa WT cells and no MET specificity in HeLa MET KO. (B) Cellular internalization of 1E7-Fc was visualized by immunofluorescent confocal microscopy. Live cells were stained with fluorescent cell plasma membrane dye (green) and treated with 1 mM of 1E7-Fc (red). Accumulation of 1E7-Fc binding is demonstrated in EBC-1 and UW-Lung-21 and moderately in HeLa MET WT. Scale bar, 20 um.

Figure 4:

1E7-Fc does not inhibit cell growth or block MET signaling pathways. (A) Proliferation assay shows that 1E7-Fc does not impact cell survival. Effect of 1E7-Fc on cell viability in vitro was assessed for indicated cell lines. Viability was assessed using Cell Counting Kit-8 cell proliferation assay 72 h after treatment with indicated concentrations of VHH-Fc. Points mean; bar SEM (n = 6). (B) Western blots were performed to assess MET, p-MET (Tyr1234/1235), AKT, p-AKT (Ser473), ERK, p-ERK (Thr202/Tyr204), and GAPDH in cells treated with 1E7-Fc. Cells were treated respective concentrations of 1E7-Fc for 4 h and Capmatinib (5 nM) was used as a positive control.

Figure 5:

1E7-Fc targets and accumulates in MET-expressing xenografts. (A) [⁸⁹Zr]Zr-1E7-Fc can detect MET-positive lung cancer xenografts by PET/CT imaging. Representative PET/CT images of mice bearing EBC-1 and UW-Lung-21 subcutaneous xenografts. Mice (n = 3 per cell line) received around 5.5 MBq [⁸⁹Zr]Zr-1E7-Fc via tail vein and imaged at the labeled time points. (B) Representative 3D PET/CT images of [⁸⁹Zr]Zr-1E7-Fc in mice bearing EBC-1 and UW-Lung-21 subcutaneous xenografts at 48 h. (C) In vivo validation of MET expression on tumor xenografts. Representative images of MET IHC staining in EBC-1 and UW-Lung-21 xenografts. Scale bar 50 μm.

Figure 6:

Confirmation of [⁸⁹Zr]Zr-1E7-Fc selectivity and stability in vivo. (A) Quantitative analysis of the same subcutaneous xenografts as Figure 5. Region of interest (ROI) were drawn to obtain values for heart and tumor for the indicated time points. Inverse relationship between the organs suggest all radiolabeled 1E7-Fc localized in the MET-positive xenograft over time. (B) Biodistribution of [⁸⁹Zr]Zr1E7-Fc in organs at 24 h. Organs and tissues are harvested at 24 h and the radioactive counts were measured for each one. There is a significantly higher uptake of [⁸⁹Zr]Zr-1E7-Fc in the tumor relative to muscle. Values are the mean (n = 3) ± SEM. (C) Biodistribution of [⁸⁹Zr]Zr-1E7-Fc at 24 h in the tumor is significantly higher than in the bone and muscle. Values are the mean (n = 3) ± SEM. (D) In vivo stability of 1E7-Fc is around three days. 1E7-Fc was injected into mice via tail vein and blood was collected at 10 time points for sera collection. The amount of 1E7-Fc remining in the sera that binds to human MET was measured with a dilution ELISA. Data are the mean (n = 3) ± SEM.