Phage Display Technology in Biomarker Identification with Emphasis on Non-Cancerous Diseases.

In recent years, phage display technology has become vital in clinical research. It helps create antibodies that can specifically bind to complex antigens, which is crucial for identifying biomarkers and improving diagnostics and treatments. However, existing reviews often overlook its importance in areas outside cancer research. This review aims to fill that gap by explaining the basics of phage display and its applications in detecting and treating various non-cancerous diseases. We focus especially on its role in degenerative diseases, inflammatory and autoimmune diseases, and chronic non-communicable diseases, showing how it is changing the way we diagnose and treat illnesses. By highlighting important discoveries and future possibilities, we hope to emphasize the significance of phage display in modern healthcare.

A synergistic therapy against influenza virus A/H1N1/PR8 by a HA1 specific neutralizing single-domain VL and an RNA hydrolyzing scFv.

The emergence of anti-influenza drug-resistant strains poses a challenge for influenza therapy due to mutations in the virus's surface protein. Recently, there has been increasing interest in combination therapy consisting of two or more drugs as a potential alternative approach, aiming to enhance therapeutic efficacy. In this study, we investigated a novel synergistic therapy with a vertical effect using a single-domain VL-HA1-specific antibody against H1N1/PR8 and a horizontal effect using an RNA catalytic antibody with broad-spectrum influenza antiviral drug. We isolated a single-domain VL-HA1-specific (NVLH8) antibody binding to the virus particles showing a neutralizing activity against influenza virus A, specifically H1N1/PR8, as determined by the reduction in plaque number and lower viral HA protein expression in vitro. The neutralizing antibody likely prevented the viral entry, specifically at the viral genome-releasing step. Additionally, the 3D8 scFv hydrolyzed viral RNAs in the cytoplasm, including mRNA, vRNA, and cRNA in MDCK cells. The combined treatment of neutralizing antibodies for a vertical effect and 3D8 scFv for a horizontal effect produced a synergistic effect providing a novel approach against viral diseases when compared with a single treatment. Our results indicated that combining treatment, in particular two proteins exhibiting different mechanisms of action increased the antiviral activity against the influenza virus.

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.

Identification and characterization of rabbit scFv antibodies suitable for immuno-affinity separation of recombinant human kynureninase from Escherichia coli cell lysate.

In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm3-bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4 %. Moreover, using the scFv column of R2-47, 90.7 % of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67 % of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E. coli cell lysate by one-step affinity purification, and 89.7 % of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

From exploring cancer and virus targets to discovering active peptides through mRNA display.

During carcinogenesis, neoplastic cells accumulate mutations in genes important for cellular homeostasis, producing defective proteins. Viral infections occur when viral capsid proteins bind to the host cell receptor, allowing the virus to enter the cells. In both cases, proteins play important roles in cancer development and viral infection, so these targets can be exploited to develop alternative treatments. mRNA display technology is a very powerful tool for the development of peptides capable of acting on specific targets in neoplastic cells or on viral capsid proteins. mRNA display technology allows the selection and evolution of peptides with desired functional properties from libraries of many nucleic acid variants. Among other advantages of this technology, the use of flexizymes allows the production of peptides with unnatural amino acid residues, which can enhance the activity of these molecules. From target immobilization, peptides with greater specificity for the targets of interest are generated during the selection rounds. Herein, we will explore the use of mRNA display technology for the development of active peptides after successive rounds of selection, using proteins present in neoplastic cells and viral particles as targets.

In vivo selection of synthetic nucleocapsids for tissue targeting.

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

Antibody Phage Display.

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.

Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning.

Phage display is a technique that allows the presentation of unique proteins on the surface of bacteriophages. The phage particles are usually screened via repetitive rounds of antigen-guided selection and phage amplification. The main advantage of this approach lies in the physical linkage between phenotype and genotype. This feature allows the isolation of single unique clones from a panning campaign consisting of a highly diverse population of clones. Due to the high-throughput nature of this technique, different approaches have been developed to assist phage display selections. One of which involves utilizing a streptavidin-coated solid-phase extraction (SPE) tip that is mounted to an electronically controlled motorized multichannel pipette. In this chapter, we will entail the procedures involved in the adaptation of a commercial SPE tip (MSIA™ streptavidin D.A.R.T's®) as the solid phase. This protocol is an updated version of a previous protocol with some minor refinements.

Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen's epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection.

Epivolve: A Protocol for Site-Directed Antibodies.

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

Progress on Phage Display Technology: Tailoring Antibodies for Cancer Immunotherapy.

The search for innovative anti-cancer drugs remains a challenge. Over the past three decades, antibodies have emerged as an essential asset in successful cancer therapy. The major obstacle in developing anti-cancer antibodies is the need for non-immunogenic antibodies against human antigens. This unique requirement highlights a disadvantage to using traditional hybridoma technology and thus demands alternative approaches, such as humanizing murine monoclonal antibodies. To overcome these hurdles, human monoclonal antibodies can be obtained directly from Phage Display libraries, a groundbreaking tool for antibody selection. These libraries consist of genetically engineered viruses, or phages, which can exhibit antibody fragments, such as scFv or Fab on their capsid. This innovation allows the in vitro selection of novel molecules directed towards cancer antigens. As foreseen when Phage Display was first described, nowadays, several Phage Display-derived antibodies have entered clinical settings or are undergoing clinical evaluation. This comprehensive review unveils the remarkable progress in this field and the possibilities of using clever strategies for phage selection and tailoring the refinement of antibodies aimed at increasingly specific targets. Moreover, the use of selected antibodies in cutting-edge formats is discussed, such as CAR (chimeric antigen receptor) in CAR T-cell therapy or ADC (antibody drug conjugate), amplifying the spectrum of potential therapeutic avenues.

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica).

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10-8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

Exploring shark VNAR antibody against infectious diseases using phage display technology.

Antibody with high affinity and specificity to antigen has widely used as a tool to combat various diseases. The variable domain of immunoglobulin new antigen receptor (VNAR) naturally found in shark contains autonomous function as single-domain antibody. Due to its excellent characteristics, the small, non-complex, and highly stable have made shark VNAR can acquires the antigen-binding capability that might not be reached by conventional antibody. Phage display technology enables shark VNAR to be presented on the surface of phage, allowing the exploration of shark VNAR as an alternative antibody format to target antigens from various infectious diseases. The application of phage-displayed shark VNAR in antibody library and biopanning eventually leads to the discovery and isolation of antigen-specific VNARs with diagnostic and therapeutic potential towards infectious diseases. This review provides an overview of the shark VNAR antibody, the types of phage display technology with comparison to the other types of display system, as well as the application and case studies of phage-displayed shark VNAR antibodies against infectious diseases.

Propagation Capacity of Phage Display Peptide Libraries Is Affected by the Length and Conformation of Displayed Peptide.

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.

Yeast biopanning against site-specific phosphorylations in tau.

The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.

Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Highly sensitive and label-free electrochemical detection of C-reactive protein on a peptide receptor-gold nanoparticle-black phosphorous nanocomposite modified electrode.

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein found in blood serum, and an enhanced CRP level is indicative of inflammatory conditions such as infection and cancer, among others. In this work, we developed a novel high CRP-affinity peptide-functionalized label-free electrochemical biosensor for the highly sensitive and selective detection of CRP. Throughout biopanning with random peptide libraries, high affinity peptides for CRP was successfully identified, and then a series of synthetic peptide receptor, of which C-terminus was incorporated to gold binding peptide (GBP) as an anchoring motif was covalently immobilized onto gold nanoparticle (AuNPs) tethered polydopamine (PDA)‒black phosphorus (BP) (AuNPs@BP@PDA) nanocomposite electrode. Interaction between the CRP-binding peptide and CRP was confirmed via enzyme-linked immunosorbent assay along with various physicochemical and electrochemical analyses. Under the optimized experimental conditions, the proposed peptide-based biosensor detects CRP in the range of 0-0.036 µg/mL with a detection limit (LOD) of 0.7 ng/mL. The developed sensor effectively detects CRP in the real samples of serum and plasma of Crohn's disease patients. Thus, the fabricated peptide-based biosensor has potential applications in clinical diagnosis and medical applications.

Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells.

Background: Glioblastoma (GBM) is both the most common and aggressive type of primary brain tumor, associated with high mortality rates and resistance to conventional therapy. Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and in vivo phage display biopanning. Methods and results: We have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining. Conclusion: These results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM.

Progress in Bioengineering of Myotropic Adeno-Associated Viral Gene Therapy Vectors.

The ability to specifically, safely, and efficiently transfer therapeutic payloads to the striated musculature via a minimally invasive delivery route remains one of the most important but also most ambitious aims in human gene therapy. Over the past two decades, a flurry of groups have harnessed recombinant adeno-associated viruses (AAVs) for this purpose, carrying cargoes that were packaged either in one of the various wild-type capsids or in a synthetic protein shell derived by molecular bioengineering. In this study, we provide an overview over the most commonly used techniques for the enrichment of muscle-specific (myotropic) AAV capsids, typically starting off with the genetic diversification of one or more extant wild-type sequences, followed by the stratification of the ensuing capsid libraries in different muscle types in small or large animals. These techniques include the shuffling of multiple parental capsid genes, peptide display in exposed capsid loops, mutagenesis of individual capsid residues, creation of chimeras between two viral parents, or combinations thereof. Moreover, we highlight alternative experimental or bioinformatic strategies such as ancestral reconstruction or rational design, all of which have already been employed successfully to derive synthetic AAV capsids or vectors with unprecedented in vivo efficiency and/or specificity in the musculature. Most recently, these efforts have culminated in the isolation of unique clades of myotropic vectors called AAVMYO or MyoAAV that have in common the display of the amino acid motif RGD (arginine-glycine-aspartate) on the capsid surface and that exhibit the highest transduction rate in striated muscles of mice or nonhuman primates reported to date. Finally, we note essential looming improvements that will facilitate and accelerate clinical translation of these latest generations of myotropic AAVs, including the identification and utilization of capsid selection or validation schemes that promise optimal translation in humans, and continued efforts to enhance patient safety by minimizing hepatic off-targeting.