Cancer Res Treat.  2019 Jul;51(3):861-875. 10.4143/crt.2018.214.

A Phage Display-Identified Peptide Selectively Binds to Kidney Injury Molecule-1 (KIM-1) and Detects KIM-1–Overexpressing Tumors In Vivo

• Haque ME
• Khan F
• Chi L
• Gurung S
• Vadevoo SM
• Park RW
• Kim DK
• Kim SK
• Lee B

• KMID: 2454279
• DOI: http://doi.org/10.4143/crt.2018.214

Abstract

PURPOSE
This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo.
MATERIALS AND METHODS
Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1-overexpressing and KIM-1-low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1-overexpressing and KIM1-low expressing tumors in mice was examined by whole-body fluorescence imaging.
RESULTS
A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1-overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1-overexpressing A498 renal tumor compared to KIM1-low expressing HepG2 liver tumor in mice.
CONCLUSION
The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1"’overexpressing tumors.

Keyword

In vivo imaging; Kidney injury molecule-1; Peptide; Phage display; Kidney neoplasms

MeSH Terms

Animals
Bacteriophages*
Clone Cells
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Fluorometry
Kidney Neoplasms
Kidney*
Liver
Lung
Mass Screening
Mice
Optical Imaging
Peptide Library
Peptides
Peptide Library
Peptides

Figure

• 

  Fig. 1. Biopanning of a phage library for peptides that selectively bind to the kidney injury molecule-1 (KIM-1) protein. (A) Schematic diagram for phage library biopanning. The phage-displayed peptide library was incubated with bovine serum albumin (BSA)–coated plates and the supernatant containing the unbound phages was then collected and incubated with KIM-1 protein-coated plates. Phages that bound to KIM-1 were eluted with low pH glycine buffer and amplified for the next round of biopanning. (B) Enrichment of phage titers during screening rounds. Five rounds of biopanning were performed and the phage titer (pfu) after each round was measured. Numbers on bars represent the enrichment fold of the phage titer at each round relative to the first round.

• 

  Fig. 2. Binding of phage clones to the kidney injury molecule-1 (KIM-1) protein and KIM-1–overexpressing cells. (A) Individual phage clones were incubated with KIM-1–coated plates and the bound phage clones were determined by enzymelinked immunosorbent assay (ELISA). Bovine serum albumin (BSA) was used as a control. Data represent the absorbance at 450 nm as the mean±standard deviation (SD) of three separate experiments performed in triplicate. (B) Immunofluorescence analysis of KIM-1 expression on cells. 769-P, A549, and HEK-293 cells were incubated with an anti–KIM-1 antibody or IgG control (red). The nucleus was stained with DAPI (blue), and images were merged with the KIM-1 staining. Scale bars=20 μm. (C) Individual phage clones were incubated with 769-P and HEK-293 cells and the bound phage clones were measured by ELISA. Data represent the absorbance at 450 nm as the mean±SD of three separate experiments performed in triplicate.

• 

  Fig. 3. Binding of peptides to the kidney injury molecule-1 (KIM-1) protein and KIM-1–overexpressing cells. (A) Binding of the CNWMINKEC and CVPSKPGLC peptides to KIM-1 protein relative to bovine serum album (BSA)–coated enzymelinked immunosorbent assay plates was determined by fluorometry. Data represent arbitrary units at 470 nm as the mean±standard deviation (SD) of three separate experiments performed in triplicate. n.s., not significant; **p < 0.01 by Students t-test. (B) 769-P and HEK-293 cells were incubated with the CNWMINKEC and CVPSKPGLC peptides labeled with FITC and were subjected to flow cytometry. Data represent the percent binding of the peptide to the cells as the mean±SD of three separate experiments. (C) Representative histograms were shown. (D) A549 and HEK-293 cells were incubated with the CNWMINKEC peptide labeled with FITC and were subjected to flow cytometry. Data represent the percent binding of the peptide to the cells as the mean±SD of three separate experiments. (E) Representative histograms were shown. n.s., not significant; **p < 0.01 by Students t-test.

• 

  Fig. 4. Binding of the CNWMINKEC peptide multimer to the kidney injury molecule-1 (KIM-1) protein and KIM-1–overexpressing cells. (A) Binding of the NeutrAvidin/biotin-CNWMINKEC peptide multimer to KIM-1 protein relative to bovine serum albumin (BSA)–coated enzyme-linked immunosorbent assay plates was determined by fluorometry. Data represent arbitrary units at 470 nm as the mean±standard deviation (SD) of three separate experiments performed in triplicate. n.s., not significant; **p < 0.01 by Students t-test. (B) 769-P and HEK-293 cells were incubated with the FITC-labeled NeutrAvidin or NeutrAvidin/biotin-CNWMINKEC peptide multimer (green) and DAPI for nuclear staining (blue). Cells were observed with an immunofluorescence microscope. Scale bars=20 μm. (C) 769-P and HEK-293 cells were incubated with the FITClabeled NeutrAvidin or NeutrAvidin/biotin-CNWMINKEC peptide multimer and subjected to flow cytometry. Data represent the percent binding of the peptide to the cells as the mean±SD of three separate experiments. n.s., not significant; **p < 0.01 by Students t-test. (D) Co-localization assays; 769-P cells were incubated with the FITC-labeled NeutrAvidin/biotin-CNWMINKEC peptide multimer (green), anti–KIM-1 antibody (red), and DAPI for nuclear staining (blue). Cells were observed under a confocal microscope. Scale bars=20 μm. (E) Competition assays; 769-P and HEK-293 cells were pre-incubated with the indicated concentrations of the anti–KIM-1 antibody and then with the NeutrAvidin/biotin-CNWMINKEC peptide multimer prior to analysis using flow cytometry. Data represent the percent binding of the peptide to the cells as the mean±SD of three separate experiments. *p < 0.05, **p < 0.01 by Students t-test.

• 

  Fig. 5. Cytotoxicity and serum stability of the CNWMINKEC peptide. (A) 769-P and HEK-293 cells were incubated with the indicated concentrations of the CNWMINKEC or control peptide for 24 hours. Cell cytotoxicity was determined using the CCK-8 assay. Data represent the mean±standard deviation of three independent experiments performed in triplicate. (B) The CNWMINKEC peptide was incubated with mouse serum at 37°C for the indicated time periods and samples were fractionated by C18 reverse-phase fast performance liquid chromatography. Samples containing peptide or serum only were used as controls. The Y axis represents milli-absorbance unit (mAU) at 215 nm. The X axis represents the retention time in minutes. The peptide peak is indicated by an arrow separable from the serum peaks.

• 

  Fig. 6. In vivo imaging and detection of tumor by the CNWMINKEC peptide. (A) Immunofluorescence analysis of kidney injury molecule-1 (KIM-1) expression. ACHN, A498 and HepG2 cells were incubated with an anti-KIM-1 antibody or IgG control (red). The nucleus was stained with DAPI (blue), and images were merged. Scale bars=20 μm. (B) Flow cytometry analysis of peptide binding to cells. ACHN, A498, and HepG2 cells were incubated with the indicated peptide labeled with FITC. Following incubation, cells were subjected to flow cytometry. Data represent the percent binding of each peptide to cells as the mean±standard deviation (SD) of three separate experiments. n.s., not significant; **p < 0.01 by Students t-test. (C) The Flamma675 NIR dye-labeled CNWMINKEC or control peptide was intravenously injected into mice bearing A498 and HepG2 tumors. In vivo whole-body fluorescence images were taken at the indicated time points post injection. Dotted circles indicate the tumor location. Scale bars indicate normalized fluorescent intensity. (D) Ex vivo fluorescence images of A498 and HepG2 tumors and other organs isolated from mice 24-hour post injection with the CNWMINKEC or control peptide. Scale bars indicate the normalized fluorescence intensity. (E) Quantification of the ex vivo fluorescence intensities of tumor and control organs in D. Data are presented as the mean±SD of intensities obtained from three mice per group. n.s., not significant; ***p < 0.001 by Students t-test. (F) Histological analysis of CNWMINKEC peptide homing to organs. The tissues in D were frozen and sections of the frozen tumor and kidney tissues were incubated with the anti–KIM-1 antibody (red) and DAPI for nuclear staining (blue). Flamma675-labeled CNWMINKEC or control peptide is shown in yellow. Scale bars=50 μm.

Reference

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