Phage display has been widely used to identify peptides binding to a variety of biological targets. In the current work, we planned to select novel peptides targeting CD4 through screening of a commercial phage display library (New England Biolabs Ph.D.^TM-7). After three rounds of biopanning, 57 phage clones were Sanger-sequenced. These clones represented 30 unique peptide sequences, which were subjected to phage ELISA, resulting in the identification of two potential target binders. Following peptide synthesis, downstream characterization was conducted using fluorescence plate-based assay, flow cytometry, SPR, and confocal microscopy. The results revealed that neither of the peptides identified in the Sanger-based phage display selection exhibited specific binding toward CD4. The naïve library and the phage pool recovered from the third round of biopanning were then subjected to next-generation sequencing (NGS). The results of NGS indicated corruption of the selection output by a phage already known as a fast-propagating clone whose target-unrelated enrichment can shed light on the misidentification of target-binding peptides through phage display. This work provides an in-depth insight into some of the challenges encountered in peptide phage display selection. Furthermore, our data highlight that NGS, by exploring a broader sequence space and providing a more precise picture of the composition of biopanning output, can be used to refine the selection protocol and avoid misleading the process of ligand identification. We hope that these findings can describe some of the complexities of phage display selection and offer help to fellow researchers who have faced similar situations.

The larger size and diversity of phage display peptide libraries enhance the probability of finding clinically valuable ligands. A simple way of increasing the throughput of selection is to mix multiple peptide libraries with different characteristics of displayed peptides and use it as biopanning input. In phage display, the peptide is genetically coupled with a biological entity (the phage), and the representation of peptides in the selection system is dependent on the propagation capacity of phages. Little is known about how the characteristics of displayed peptides affect the propagation capacity of the pooled library. In this work, next-generation sequencing (NGS) was used to investigate the amplification capacity of three widely used commercial phage display peptide libraries (Ph.D.™-7, Ph.D.™-12, and Ph.D.™-C7C from New England Biolabs). The three libraries were pooled and subjected to competitive propagation, and the proportion of each library in the pool was quantitated at two time points during propagation. The results of the inter-library competitive propagation assay led to the conclusion that the propagation capacity of phage libraries on a population level is decreased with increasing length and cyclic conformation of displayed peptides. Moreover, the enrichment factor (EF) analysis of the phage population revealed a higher propagation capacity of the Ph.D.^TM-7 library. Our findings provide evidence for the contribution of the length and structural conformation of displayed peptides to the unequal propagation rates of phage display libraries and suggest that it is important to take peptide characteristics into account once pooling multiple combinatorial libraries for phage display selection through biopanning.

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.