Phage Display in Cancer Research: Special Issue Editorial.

Soon after its birth in 1985, following a short lag period [...].

Discovery of a new class of cell-penetrating peptides by novel phage display platform.

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library.

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

In vivo phage display identifies novel peptides for cardiac targeting.

Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice.

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

Identification of a potent PCNA-p15-interaction inhibitor by autodisplay-based peptide library screening.

Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay-based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12-mer randomized peptide library consisting of 2.54 × 106 colony-forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet-I-PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet-I-PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 µM for PCNA-P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP-box containing protein involved in DNA replication and repair. P3 inhibited the PCNA-p1551-70 interaction with a half maximal inhibitory activity of 16.2 µM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.

Improving Pharmacokinetics of Peptides Using Phage Display.

Phage display is a versatile method often used in the discovery of peptides that targets disease-related biomarkers. A major advantage of this technology is the ease and cost efficiency of affinity selection, also known as biopanning, to identify novel peptides. While it is relatively straightforward to identify peptides with optimal binding affinity, the pharmacokinetics of the selected peptides often prove to be suboptimal. Therefore, careful consideration of the experimental conditions, including the choice of using in vitro, in situ, or in vivo affinity selections, is essential in generating peptides with high affinity and specificity that also demonstrate desirable pharmacokinetics. Specifically, in vivo biopanning, or the combination of in vitro, in situ, and in vivo affinity selections, has been proven to influence the biodistribution and clearance of peptides and peptide-conjugated nanoparticles. Additionally, the marked difference in properties between peptides and nanoparticles must be considered. While peptide biodistribution depends primarily on physiochemical properties and can be modified by amino acid modifications, the size and shape of nanoparticles also affect both absorption and distribution. Thus, optimization of the desired pharmacokinetic properties should be an important consideration in biopanning strategies to enable the selection of peptides and peptide-conjugated nanoparticles that effectively target biomarkers in vivo.

Sequence-based design and construction of synthetic nanobody library.

Nanobody (Nb), the smallest antibody fragments known to bind antigens, is now widely applied to various studies, including protein structure analysis, bioassay, diagnosis, and biomedicine. The traditional approach to generating specific nanobodies involves animal immunization which is time-consuming and expensive. As the understanding of the antibody repertoire accumulation, the synthetic library, which is devoid of animals, has attracted attention widely in recent years. Here, we describe a synthetic phage display library (S-Library), designed based on the systematic analysis of the next-generation sequencing (NGS) of nanobody repertoire. The library consists of a single highly conserved scaffold (IGHV3S65*01-IGHJ4*01) and complementary determining regions of constrained diversity. The S-Library containing 2.19 × 108 independent clones was constructed by the one-step assembly and rapid electro-transformation. The S-Library was screened against various targets (Nb G8, fusion protein of Nb G8 and green fluorescent protein, bovine serum albumin, ovalbumin, and acetylcholinesterase). In comparison, a naïve library (N-Library) from the source of 13 healthy animals was constructed and screened against the same targets as the S-Library. Binders were isolated from both S-Library and N-Library. The dynamic affinity was evaluated by the biolayer interferometry. The data confirms that the feature of the Nb repertoire is conducive to reducing the complexity of library design, thus allowing the S-Library to be built on conventional reagents and primers.

Preparation of Bioconjugates of Chimeric M13 Phage and Gold Nanorods.

Phage-nanomaterial conjugates are functional bio-nanofibers with various applications. While phage display can select for phages with desired genetically encoded functions and properties, nanomaterials can endow the phages with additional features at nanoscale dimensions. Therefore, combining phages with nanotechnology can construct bioconjugates with unique characteristics. One strategy for filamentous phages is to adsorb nanoparticles onto the side wall, composed of pVIII subunits, through electrostatic interactions. However, a noncovalent approach may cause offloading if the environment changes, potentially causing side effects especially for in vivo applications. Therefore, building stable phage-bioconjugates is an important need. We previously reported the construction of chimeric M13 phage conjugated with gold nanorods, named "phanorods," without weakening the binding affinity to the bacterial host cells. Herein, we give a detailed protocol for preparing the chimeric M13 phage and covalently conjugating gold nanorods to the phage.

Exploring the short linear motif-mediated protein-protein interactions of CrkL through ProP-PD.

Adaptor proteins play a pivotal role in cellular signaling mediating a multitude of protein-protein interaction critical for cellular homeostasis. Dysregulation of these interactions has been linked to the onset of various cancer pathologies and exploited by viral pathogens during host cell takeover. CrkL is an adaptor protein composed of an N-terminal SH2 domain followed by two SH3 domains that mediate interactions with diverse partners through the recognition of specific binding motifs. In this study, we employed proteomic peptide-phage display (ProP-PD) to comprehensively explore the short linear motif (SLiM)-based interactions of CrkL. Furthermore, we scrutinized how the binding affinity for selected peptides was influenced in the context of the full-length CrkL versus the isolated N-SH3 domain. Importantly, our results provided insights into SLiM-binding sites within previously reported interactors, as well as revealing novel human and viral ligands, expanding our understanding of the interactions mediated by CrkL and highlighting the significance of SLiM-based interactions in mediating adaptor protein function, with implications for cancer and viral pathologies.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers.

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Developing recombinant antibodies by phage display technology to neutralize viral infectious diseases.

The use of recombinant antibodies developed through phage display technology offers a promising approach for combating viral infectious diseases. By specifically targeting antigens on viral surfaces, these antibodies have the potential to reduce the severity of infections or even prevent them altogether. With the emergence of new and more virulent strains of viruses, it is crucial to develop innovative methods to counteract them. Phage display technology has proven successful in generating recombinant antibodies capable of targeting specific viral antigens, thereby providing a powerful tool to fight viral infections. In this mini-review article, we examine the development of these antibodies using phage display technology, and discuss the associated challenges and opportunities in developing novel treatments for viral infectious diseases. Furthermore, we provide an overview of phage display technology. As these methods continue to evolve and improve, novel and sophisticated tools based on phage display and peptide display systems are constantly emerging, offering exciting prospects for solving scientific, medical, and technological problems related to viral infectious diseases in the near future.

Massively-multiplexed epitope mapping techniques for viral antigen discovery.

Following viral infection, viral antigens bind specifically to receptors on the surface of lymphocytes thereby activating adaptive immunity in the host. An epitope, the smallest structural and functional unit of an antigen, binds specifically to an antibody or antigen receptor, to serve as key sites for the activation of adaptive immunity. The complexity and diverse range of epitopes are essential to study and map for the diagnosis of disease, the design of vaccines and for immunotherapy. Mapping the location of these specific epitopes has become a hot topic in immunology and immune therapy. Recently, epitope mapping techniques have evolved to become multiplexed, with the advent of high-throughput sequencing and techniques such as bacteriophage-display libraries and deep mutational scanning. Here, we briefly introduce the principles, advantages, and disadvantages of the latest epitope mapping techniques with examples for viral antigen discovery.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Streamlined Data Analysis Pipeline for Deep Sequence-Coupled Biopanning Identification of Pathogen-Specific Antibody Responses in Serum.

Deep sequence-coupled biopanning (DSCB) is a powerful tool that couples affinity selection of a bacteriophage MS2 virus-like particle peptide display platform with deep sequencing. While this approach has been used successfully to investigate pathogen-specific antibody responses in human sera, data analysis is time-consuming and complicated. Here, we describe a streamlined data analysis method for DSCB using MATLAB, expanding the potential for this approach to be deployed rapidly and consistently.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

Rapid Evaluation of Staple Placement in Stabilized α Helices Using Bacterial Surface Display.

There are a wealth of proteins involved in disease that cannot be targeted by current therapeutics because they are inside cells, inaccessible to most macromolecules, and lack small-molecule binding pockets. Stapled peptides, where two amino acids are covalently linked, form a class of macrocycles that have the potential to penetrate cell membranes and disrupt intracellular protein-protein interactions. However, their discovery relies on solid-phase synthesis, greatly limiting queries into their complex design space involving amino acid sequence, staple location, and staple chemistry. Here, we use stabilized peptide engineering by Escherichia coli display (SPEED), which utilizes noncanonical amino acids and click chemistry for stabilization, to rapidly screen staple location and linker structure to accelerate peptide design. After using SPEED to confirm hotspots in the mdm2-p53 interaction, we evaluated different staple locations and staple chemistry to identify several novel nanomolar and sub-nanomolar antagonists. Next, we evaluated SPEED in the B cell lymphoma 2 (Bcl-2) protein family, which is responsible for regulating apoptosis. We report that novel staple locations modified in the context of BIM, a high affinity but nonspecific naturally occurring peptide, improve its specificity against the highly homologous proteins in the Bcl-2 family. These compounds demonstrate the importance of screening linker location and chemistry in identifying high affinity and specific peptide antagonists. Therefore, SPEED can be used as a versatile platform to evaluate multiple design criteria for stabilized peptide engineering.

Concerted Antibody and Antigen Discovery by Differential Whole-cell Phage Display Selections and Multi-omic Target Deconvolution.

Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.

Screening of novel peptides that specifically interact with vitamin D bound biocomplex proteins.

The majority of the vitamin D that is present in the blood binds to vitamin D binding protein (VDBP) and circulates in the form of a complex (VDBP-Complex). Knowing the level of vitamin D in the body is crucial for vitamin D-related treatments so that the right dosage of vitamin D can be given. In other words, it is essential to distinguish between the protein VDBP and the complex form bound to vitamin D. As a novel way for the detection of VDBP-Complex, a more effective phage display methodology was applied in this study along with the addition of two approaches. In order to screen a sequence specific to the target only, the pre-binding method and after-binding method were performed. VDBP-Complex was directly coated on the petri dishes. In order to select phages that specifically bind to the VDBP-Complex, random phages were attached, and selected by 7 times of biopanning. Individual DNA sequences were analyzed for each biopanning to find specific peptide sequences for VDBP-Complex. The affinity of binding phages was verified by ELISA assay using an anti-M13 antibody. The phage having a sequence of SFTKTSTFTWRD (called as M3) has shown the highest binding affinity to VDBP-Complex. As a result of the removal test of VDBP-Complex using magnetic beads conjugated with M3 peptide, it was confirmed that significant decrease of VDBP-Complex. The unique characteristic of the M3 sequence was confirmed through a sequence-modified peptide (SFT motif). That is, it is expected that the M3 peptide may be used to determine the vitamin D levels in the blood.

Recombinant antibodies by phage display for bioanalytical applications.

Antibody phage display, aimed at preparing antibodies to defined antigens, is a useful replacement for hybridoma technology. The phage system replaces all work stages that follow animal immunization with simple procedures for manipulating DNA and bacteria. It enables the time needed to generate stable antibody-producing clones to be shortened considerably, making the process noticeably cheaper. Antibodies prepared by phage display undergo several affinity selection steps and can be used as selective receptors in biosensors. This article briefly describes the techniques used in the making of phage antibodies to various antigens. The possibilities and prospects are discussed of using phage antibodies as selective agents in analytical systems, including biosensors.