Phage Display Research Articles (Page 1)

A synthetic humanized camelid single-domain antibody targeting VEGF exhibits in vitro antiangiogenic effects.

In colorectal cancer (CRC), the upregulation of Candida albicans (C. albicans) abundance significantly promotes tumor progression and exacerbates immunosuppression. In this study, we demonstrate that candidalysin-induced macrophage pyroptosis contributes to the immunosuppressive tumor microenvironment (TME). Conversely, the neutralizing peptide of candidalysin, isolated via phage display, effectively protects macrophages from pyroptosis. Based on this, we constructed an epigenetic inhibitor-loaded nanomodulator to target the interaction between C. albicans and macrophages. To ensure precise targeting of intraspecific morphological differences, the nanomodulator is covered with the C. albicans pretreated macrophage membranes. The nanomodulator exhibits the ability to protect macrophages from pyroptosis and reprograms the metabolic and immune response of macrophages, while the epigenetic inhibitor upregulates tumor self-antigen presentation. In vivo, the nanomodulator has been shown to effectively sustain macrophage activation within the TME and promotes a robust IL-17-mediated immune response. Meanwhile, the nanomodulator exhibited excellent immune memory effects and effectively synergized with immune checkpoint blockade therapy in an orthotopic CRC model. This approach of manipulating the C. albicans-macrophage crosstalk to augment therapeutic efficacy in CRC offers promising insights for the clinical management of CRC.

The human immune system evolved to defend against the panoply of microbial threats. By harnessing such ability, vaccines have cumulatively saved hundreds of millions of lives. Despite such tremendous success, there have also been remarkable failures, such as the lack of a clinically proven vaccine against Staphylococcus aureus (SA), which continues to pose an urgent public health threat. In practice, it has proven challenging to identify the molecular basis for relevant epitopes for this pathogen. Here, we summarize our experience implementing an integrated approach using phage display technology for the identification of B-cell epitopes of microbial virulence factors, which we developed with a focus on SA. This approach was used to define minimal B-cell epitopes of the staphylococcal leucocidin family of pore-forming toxins (PFTs) that have been implicated in staphylococcal clinical infection. Our methodology provides proof of principle for an approach well suited for the rapid and efficient generation of modular protein-based vaccines for protection from clinical infection, which can be used to target pathogens for which no vaccine is currently available.

To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein-protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody-epitope binding interactions.

Capsid structure of phage SPO1 reveals novel minor capsid proteins and insights into capsid stabilization.

Streptococcus agalactiae (GBS) has emerged as one of the most prevalent bacterial pathogens causing severe economic losses in tilapia aquaculture due to its highly contagious and lethal nature. Nanobodies (Nbs), characterized by their small molecular size, enhanced tissue penetration, high tolerance, and exceptional antigen-binding affinity, represent a promising green alternative to conventional antibiotics. In the present study, the objective was to explore the potential of specific Nbs in the treatment of tilapia GBS disease. We first screened specific Nbs targeting the surface immunogenic (Sip) protein of GBS from a naïve phage display library, and a novel nanobody Nb30 was obtained. Nb30 was expressed in Escherichia coli and purified using the Ni-NTA Agarose column. Indirect ELISA showed that Nb30 had a high affinity against Sip and GBS in vitro. Moreover, Nb30 significantly reduced GBS colonization in the liver, spleen, and brain of GBS-infected tilapia. The survival rate in the control groups was 53%, whereas it was increased to 86% after treatment with 100 mg/kg Nb30. Transcriptome profiling revealed that Nb30 could modulate critical biological processes, including antioxidant defense, immune regulation, amino acid/protein synthesis, and energy metabolism in the liver tissues of GBS-infection tilapia. Notably, the expression levels of antioxidant enzymes (cat and gpx) were significantly up-regulated, and the TLR/MyD88/NF-κB pathway-related genes (tlr5, myd88, irak4, traf6, Rela, and NF-κB2) were significantly down-regulated after treatment with Nb30. Collectively, these findings establish a novel therapeutic strategy for controlling GBS infection in tilapia and provide evidence supporting the application of nanobodies as sustainable alternatives to antibiotics in aquaculture disease management.

We report a chemoenzymatic strategy for generating graspeptide mimetics. This strategy exploits the compatibility of sortase with the ester group at the X position of the pentapeptide sequence (LPXTG). Six types of graspeptide mimetics, including monocyclic and bicyclic depsipeptides, were generated. Combined with phage display, we identified a bicyclic depsipeptide targeting TEAD4 with a KD value of 2.2 μM. This study demonstrates the potential of peptiligase-based system for constructing cyclic peptide libraries with unique topologies.

Antibody-based radiotheranostics hold significant promise for tumor diagnosis and treatment; however, the clinical translation of radiotherapeutic agents is significantly hindered by suboptimal pharmacokinetics, particularly undesirable retention in healthy tissues. In this study, we developed novel high-affinity anti-Trop2 nanobodies using phage display technology and then generated two VHH-Fc constructs, B9 and C10, which are recombinant single-chain antibodies fused with IgG1-Fc. These were radiolabeled with [99mTc]Tc and [177Lu]Lu. A systematic comparison was then conducted against the clinically relevant monoclonal antibody hRS7 (sacituzumab). The [99mTc]Tc-labeled-B9 exhibited high Trop2-binding affinity, rapid tumor accumulation, and substantially decreased hepatic and renal uptake in BxPC-3 xenograft models compared with [99mTc]Tc-hRS7. Comprehensive SPECT/CT imaging and quantitative biodistribution studies further corroborated its prolonged intratumoral retention. When radiolabeled with [177Lu]Lu, [177Lu]Lu-B9 maintained robust tumor uptake while demonstrating faster clearance from nontarget tissues than the monoclonal antibody (hRS7). Quantitative analysis over 168 h postinjection revealed a 56.17% reduction in hepatic uptake and a 23.68% decrease in renal uptake compared to [177Lu]Lu-hRS7. In a preclinical mouse model, two-cycle administration of [177Lu]Lu-B9 induced significant tumor growth inhibition, prolonged survival, and minimal systemic toxicity. These findings highlight VHH-Fc-based radiotheranostics as a promising strategy to achieve favorable tumor retention while reducing off-target organ exposure.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inflicts enormous economic losses on the global swine industry and imposes significant pressure on agricultural production. However, there are currently no clinically approved effective therapeutics specifically targeting PRRSV. Accordingly, the development of novel antiviral agents against PRRSV is urgently needed. Notably, the structural glycoprotein 4 (GP4) of PRRSV—which plays a crucial role in viral entry into host cells—represents a promising target for antiviral development. Nanobodies, characterized by their small size, structural stability, high affinity, and excellent solubility, have emerged as attractive candidates for next-generation therapeutic development. Yet, to date, no specific nanobodies targeting PRRSV GP4 have been reported. In this study, we isolated GP4-specific nanobodies using phage display technology and investigated their mechanisms underlying viral suppression through a series of in vitro functional assays. Our results demonstrate that Nb6, Nb31, and Nb85 significantly inhibit PRRSV infection by disrupting both viral attachment to host cells and subsequent internalization processes. Collectively, these findings indicate that Nb6, Nb31, and Nb85 hold substantial potential for development as antiviral agents against PRRSV infection.

Pathogen biosensors for food safety and public healthcare based on specific biological probes biopanned by phage display: A critical review.

Phage display screening for highly specific nickel- and cobalt-binding peptides for bio-recovery of metals.

We report development and characterization of small non-immunoglobulin affibody affinity proteins directed to the highly glycosylated human carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5, CEA), and their use in immunohistochemical (IHC) analyses of human pancreatic cancer samples and for in vivo tumor imaging. A total of nineteen unique anti-CEA affibodies were identified from large phage display libraries constructed using combinatorial protein engineering of a small 58 amino acid three-helix bundle protein domain. Molecular modeling suggested that all enriched clones share a binding surface with several clustered tryptophan residues interacting with a hydrophobic patch in the N1 domain of CEA centered around a phenylalanine residue. One variant, designated as C9, exhibited the highest affinity in biosensor analyses and was reformatted into a 15 kDa homodimer expressed in Escherichia coli. The biotinylated form, C9-C9-Bio, was evaluated for its IHC performance on matched frozen and formalin-fixed, paraffin-embedded (FFPE) sections of human pancreatic cancer samples (n = 7). Compared to clinical-grade monoclonal antibodies II-7 and CEA31, as well as a polyclonal reagent, C9-C9-Bio demonstrated highly sensitive CEA detection with minimal background staining. Statistical analyses including intraclass correlation and Bland-Altman assessments revealed excellent agreement between C9-C9-Bio and the two monoclonal antibodies in FFPE tissue samples. Further, a 99mTc[Tc]-labeled C9-C9 construct showed CEA-dependent binding to human cancer cell lines in vitro, and selectively bound to CEA-expressing BxPC3 xenografts in mice when investigated as a tracer for in vivo imaging, allowing for a visualization of tumors after four hours. In summary, these findings highlight the potential use of the easily produced CEA-binding C9 affibody for various clinical applications, including IHC and medical imaging, and as a targeting moiety for directing various therapeutic modalities to CEA-expressing tumors.

Fluorescent Probes Targeting Acyl-CoA Synthetase Long-Chain Family Member 4 for Intraoperative Imaging of Hepatocellular Carcinoma.

Monomeric structure of influenza A virus NEP/NS2 obtained with an artificial protein highlights conformational plasticity.

Each year, snakebite envenoming claims thousands of lives and causes severe injury to victims across sub-Saharan Africa, many of whom depend on antivenoms derived from animal plasma as their sole treatment option1. Traditional antivenoms are expensive, can cause adverse immunological reactions, offer limited efficacy against local tissue damage and are often ineffective against all medically relevant snake species2. There is thus an urgent unmet medical need for innovation in snakebite envenoming therapy. However, developing broad-spectrum treatments is highly challenging owing to the vast diversity of venomous snakes and the complex and variable composition of their venoms3. Here we addressed this challenge by immunizing an alpaca and a llama with the venoms of 18 different snakes, including mambas, cobras and a rinkhals, constructing phage display libraries, and identifying high-affinity broadly neutralizing nanobodies. We combined eight of these nanobodies into a defined oligoclonal mixture, resulting in an experimental polyvalent recombinant antivenom that was capable of neutralizing seven toxin families orsubfamilies. This antivenom effectively prevented venom-induced lethality in vivo across 17 African elapid snake species and markedly reduced venom-induced dermonecrosis for all tested cytotoxic venoms. The recombinant antivenom performed better than a currently used plasma-derived antivenom and therefore shows considerable promise for comprehensive, continent-wide protection against snakebites by all medically relevant African elapids.

Monoclonal antibodies (mAbs) represent one of the most successful classes of biopharmaceuticals, with more than 100 approved for treating oncological, immunological, and infectious diseases. Antibody discovery and development have been driven by diverse methodologies. Classical strategies such as mouse hybridoma technology, phage display, transgenic mouse models, and single B cell isolation have enabled the generation of high-affinity therapeutic antibodies. Beyond binding affinity, recent innovations in combinatorial antibody libraries have facilitated the selection of functional antibodies within cellular environments, revealing their ability to act as agonists or antagonists and influence signal transduction pathways. These insights expand therapeutic applications by enabling modulation of complex cellular responses. Recent breakthroughs in artificial intelligence, involving antibody generation supported by rapidly growing antibody sequence and structure databases, are transforming computational protein design. This review highlights five major approaches (hybridoma technology, phage display, transgenic mouse models, and single B cell isolation, de novo antibody design) for antibody discovery and development. These approaches offer innovative strategies designed to accelerate the discovery process and enhance therapeutic outcomes for human diseases.

c-Myc is an oncogene that is dysregulated in ∼70% of cancers. Its multifaceted function complicates effective drug targeting of the protein. i-Motif (iM) DNA structures in gene promoter regions have gained attention for their potential role in the modulation of gene expression. These include the iM formed by the cytosine-rich sequence which lies upstream of the key P1 promoter of the c-Myc gene. Currently, selective ligands interacting with iM structures are limited. Here, peptide ligands for the iM from the promoter of c-Myc were identified via phage display. Hit peptides were filtered for selective binding to iM structures over other DNA structures using displacement assays and DNA melting experiments. Two lead peptides were found to produce dose-dependent changes in c-Myc gene expression after delivery into HEK293 cells expressing a c-Myc luciferase reporter construct. These leads may be used as chemical tools for the manipulation of c-Myc iMin vitro and have the potential to be developed into cell-permeable peptidomimetics for deliveryin vivo.

Combination vaccines promise to simplify immunization schedules and improve coverage, but remain technically challenging owing to antigen compatibility, immunogenic balance and formulation complexity. Here we report a modular strategy that uses a single-component nanobody binder to noncovalently attach diverse antigens to intact particles from the licensed hepatitis E vaccine. To identify a suitable binder, an alpaca was immunized with the vaccine, and nanobodies were screened via phage display. One nanobody, P1-5B, selectively bound recessed, non-immunodominant sites on the particle surface and enabled stable antigen display without disrupting native immunogenicity. Using this binder, we generated three vaccine formulations displaying five to eleven antigens, including variants from SARS-2 coronavirus, influenza virus and respiratory syncytial virus. These multivalent particles exhibited high-affinity assembly, preserved solubility and induced neutralizing titres up to three log units higher than soluble antigens. In mice, hamsters and non-human primates, the candidate vaccines conferred robust protection and showed a favourable safety profile. This approach introduces a scalable, plug-and-display system for rapid development of customizable combination vaccines.

The GDF15-GFRaL-RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15-GFRaL protein-protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.

Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display "libraries," comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions-all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance-generically called the "selector"-to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.