Identification of Urokinase Plasminogen Activator Receptor (uPAR)-Binding Peptides Using Phage Display: Strategy and Downstream Validation
5 affiliations
- 1Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital—Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
- 2Department of Chemistry, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
- 3Department of Otorhinolaryngology, Head & Neck Surgery and Audiology, Copenhagen University Hospital-Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark
- 4MDxCORE, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, 2100 Copenhagen, Denmark
- 5Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
Abstract
Background: Peptides that selectively bind to cancer-associated proteins are attractive candidates for molecular imaging and targeted therapeutic applications. The urokinase plasminogen activator receptor (uPAR) represents a promising molecular target as it is highly expressed in several cancers and has been linked to cancer aggressiveness, invasion, and metastasis. This study applied an NGS-informed biopanning strategy against uPAR, combined with multi-assay downstream validation, to identify and prioritize candidate peptides. Methods: An optimized biopanning protocol incorporating additional selection controls was combined with next-generation sequencing to identify candidate uPAR-binding peptides from a phage display library. Selected peptides were evaluated using complementary in vitro assays, including flow cytometry and a quantitative plate assay. Results: Three peptide candidates were identified and selected for further evaluation with complementary in vitro assays. The optimized workflow incorporates measures to reduce, but not eliminate, enrichment of target-unrelated peptides, and includes complementary validation of the peptides in in vitro assays. Differences observed between the validation methods highlighted the importance of evaluating peptide candidates using complementary assays. Conclusions: This study demonstrates the value of combining an optimized phage display selection with next-generation sequencing and complementary validation assays for identifying candidate peptides. The identified peptides represent promising candidates for further optimization toward molecular imaging and targeted therapeutic applications.
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BibTeX
@article{sloth2026identification,
title = {{Identification of Urokinase Plasminogen Activator Receptor (uPAR)-Binding Peptides Using Phage Display: Strategy and Downstream Validation}},
author = {Ane Beth Sloth and Ida Melisa Dawoodi and Tuule Treiberg and Anders Wilgaard Sinkjaer and Ida Vang Andersen and Amanda Oester Andersen and Emilie Caroline Skuladottir Bøgestad and Maria Rossing and Camilla Stavnsbjerg and Babak Bakhshinejad and Andreas Kjaer},
journal = {Cancers},
year = {2026},
month = oct,
doi = {10.3390/cancers18203380},
url = {https://www.mdpi.com/2072-6694/18/20/3380},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Identification of Urokinase Plasminogen Activator Receptor (uPAR)-Binding Peptides Using Phage Display: Strategy and Downstream Validation
AU - Ane Beth Sloth
AU - Ida Melisa Dawoodi
AU - Tuule Treiberg
AU - Anders Wilgaard Sinkjaer
AU - Ida Vang Andersen
AU - Amanda Oester Andersen
AU - Emilie Caroline Skuladottir Bøgestad
AU - Maria Rossing
AU - Camilla Stavnsbjerg
AU - Babak Bakhshinejad
AU - Andreas Kjaer
PY - 2026
DA - 2026/10/01
JO - Cancers
DO - 10.3390/cancers18203380
UR - https://www.mdpi.com/2072-6694/18/20/3380
AB - Background: Peptides that selectively bind to cancer-associated proteins are attractive candidates for molecular imaging and targeted therapeutic applications. The urokinase plasminogen activator receptor (uPAR) represents a promising molecular target as it is highly expressed in several cancers and has been linked to cancer aggressiveness, invasion, and metastasis. This study applied an NGS-informed biopanning strategy against uPAR, combined with multi-assay downstream validation, to identify and prioritize candidate peptides. Methods: An optimized biopanning protocol incorporating additional selection controls was combined with next-generation sequencing to identify candidate uPAR-binding peptides from a phage display library. Selected peptides were evaluated using complementary in vitro assays, including flow cytometry and a quantitative plate assay. Results: Three peptide candidates were identified and selected for further evaluation with complementary in vitro assays. The optimized workflow incorporates measures to reduce, but not eliminate, enrichment of target-unrelated peptides, and includes complementary validation of the peptides in in vitro assays. Differences observed between the validation methods highlighted the importance of evaluating peptide candidates using complementary assays. Conclusions: This study demonstrates the value of combining an optimized phage display selection with next-generation sequencing and complementary validation assays for identifying candidate peptides. The identified peptides represent promising candidates for further optimization toward molecular imaging and targeted therapeutic applications.
SN - 2072-6694
ER -