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In vivo phage display biopanning. The in vivo screening of phage display library is carried out through multiple consecutive steps which include intravenous administration of the library into the mouse, circulation of the library in the body, animal sacrifice, tissue extraction, recovery of tissue-bound phages, amplification and, finally, sequencing of the recovered phages
Source publication
Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in t...
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The objective of this study was isolation of humanized single-chain variable fragment (scFv) antibodies against IL1RAP, a promising cancer marker in leukemia and other tumors. Here, we screened for antibody fragments that recognized IL1RAP using a phage-display library of humanized scFv through several rounds of bio-panning and further FACS selecti...
The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.
Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides (including protease inhi...
Purpose
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Background:
The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which...
Citations
... Traditionally, the analysis of phage display experimental results involves sequencing a limited number of individual phage clones to identify candidate peptides [13][14][15][16] . This approach leads to loss of information and restricts the diversity of discovered target-binding peptide populations. ...
... Several factors could contribute to this outcome. One possibility is the involvement of technical aspects, such as sample loss during phage amplification steps or difficulties in retrieving phages from the tissues 15,16 . This makes a great limitation to the applied enrichment analysis as normalisation cannot account for the missing values. ...
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.
... In this process, phages displaying VHHs with greater target specificity are selected. Two different methods are commonly used for antigen immobilization on solid supports: passive adsorption and biotin conjugation (50). Passive adsorption involves directly immobilizing the antigen onto the support without the addition of chemical substances that may interfere with antigen binding. ...
... The newly infected cells produce phages displaying VHHs with higher specificity compared to the previous cycle. This amplification process leads to the enrichment of binding phages in each cycle (50). Several cycles of biopanning help reduce the variability of ligands and select for a specific subpopulation. ...
... Several cycles of biopanning help reduce the variability of ligands and select for a specific subpopulation. However, a balance must be maintained in the number of cycles to avoid excluding viable ligands or selecting non-specific antibodies (50). Among the selected materials, 3 and 4 cycles of biopanning were the most used, aiming to obtain specific Nbs without excluding potential ligands. ...
Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry.
... The loss of function of some phage may also be associated with an effect on multiplication, i.e., the phage with a high propagation rate has an advantage. This behavior is a result of the introduction of bias in the library since changes in the peptide composition and motif frequency occur every time the phages are amplified in E. coli host cells (Bakhshinejad et al., 2016). The relation between frequency and strength can be affected due to the loss of a small number of particles during the consecutive washing steps , or due to the phage clone adsorption to plastic surfaces or albumin, which is not so easy to prevent (Menendez & Scott, 2005;Vodnik et al., 2011). ...
Yttrium is a heavy rare earth element (REE) that acquires remarkable characteristics when it is in oxide form and doped with other REEs. Owing to these characteristics Y2O3 can be used in the manufacture of several products. However, a supply deficit of this mineral is expected in the coming years, contributing to its price fluctuation. Thus, developing an efficient, cost‐effective, and eco‐friendly process to recover Y2O3 from secondary sources has become necessary. In this study, we used phage surface display to screen peptides with high specificity for Y2O3 particles. After three rounds of enrichment, a phage expressing the peptide TRTGCHVPRCNTLS (DM39) from the random pVIII phage peptide library Cys4 was found to bind specifically to Y2O3, being 531.6‐fold more efficient than the wild‐type phage. The phage DM39 contains two arginines in the polar side chains, which may have contributed to the interaction between the mineral targets. Immunofluorescence assays identified that the peptide's affinity was strong for Y2O3 and negligible to LaPO4:Ce³⁺,Tb³⁺. The identification of a peptide with high specificity and affinity for Y2O3 provides a potentially new strategic approach to recycle this type of material from secondary sources, especially from electronic scrap.
... These materials exhibit different physical or chemical properties depending on the direction or orientation of their structure. For example, some directional materials can selectively bind to specific targets [14][15][16], enhance drug delivery or modulate immune responses. The design and synthesis of directional materials require precise control over their shape, size, composition, and surface functionality [17]. ...
In the realm of science and medicine, nanotechnology emerges as a beacon of hope, promising to revolutionize healthcare with its ability to manipulate matter at the molecular level. As a passionate advocate for this field, we have witnessed firsthand the transformative potential of nanoscale innovations. The promise of nanotechnology lies not only in its current applications but also in its vast, untapped potential to address some of the most pressing medical challenges of our time. Nanotechnology is a branch of science and medicine that explores the possibilities of manipulating matter at the molecular scale. Nanotechnology has already demonstrated its usefulness in various fields and has enormous, untapped potential to address some of the most pressing medical challenges of our era. However, nanotechnology also poses some risks that need to be carefully assessed by toxicology studies on nanomedicines. Nanotechnology offers many benefits for the development of new drugs and delivery systems, but it also has some potential drawbacks that require careful evaluation by toxicological studies on nanomaterials. These studies aim to identify and quantify the possible adverse effects of nanotechnology on human health and the environment, as well as to guide the safe and ethical use of nanomedicines. Nanotechnology has the potential to improve the lives of millions of people around the world, especially in developing countries where access to health care and other resources is limited. However, to achieve this goal, we need to create a culture of collaboration and transparency among all the stakeholders involved in the development and application of nanotechnology. This includes scientists, doctors, policymakers, and the public. By sharing knowledge, data, and best practices, we can ensure that nanotechnology is used ethically, safely, and effectively for the common good. Overcoming Barriers Despite the advancements, the journey of nanotechnology from the lab bench to the bedside is fraught with barriers. Regulatory hurdles, public perception, and a lack of interdisciplinary collaboration often slow the pace of progress. It is imperative that we, as a scientific community, work together to overcome these obstacles. By fostering an environment of open communication and cooperation between researchers, clinicians, and policymakers, we can ensure that the benefits of nanotechnology reach those in need. Nanomaterials are very small particles that have unique properties and applications in medicine, engineering, and other fields. However, before they can be used safely and effectively in humans or animals, they need to undergo rigorous testing to evaluate their biocompatibility. This means that they should not interfere with the normal functions of living tissues, cells, and molecules, or cause any adverse effects or toxicity. Biocompatibility testing is essential for ensuring the safety and efficacy of nanomaterials in clinical settings, where they can be used for diagnosis, treatment, or prevention of diseases. Nanotechnology has made significant progress in various fields of medicine, such as drug delivery, imaging, diagnosis, and therapy. However, there are still many challenges and obstacles that hinder the translation of nanotechnology from the laboratory to the clinic. One of the major hurdles is the evaluation of the safety and efficacy of nanomaterials in biological systems, both in vivo and in vitro. These assessments are crucial for ensuring the biocompatibility, functionality, and performance of nanomaterials in clinical applications. These tests are essential for ensuring that nanomaterials are compatible with living tissues, cells, and molecules and that they can perform their intended functions without causing harm or side effects in clinical settings.
... Phage display peptide libraries have shown great potential in identifying ligands specific to a target. However, there is a risk of falsepositive results due to the possibility of low affinity peptides being identified during the biopanning process (Bakhshinejad et al., 2016). To ensure the reliability of the screening results, it is crucial to assess the affinity of the identified peptides to their targets. ...
... Sequence analysis and structural prediction of phage-derived peptides must also be carefully considered, as well as the possibility of supramolecular assembly of the peptide Zhou et al., 2023). False positives are also a challenge in phage display screening (Bakhshinejad et al., 2016), with many phages already occupied by non-specific adsorption before positive selection. Considering that phages bind to plastic substrates or sealers (Lamboy et al., 2009), a large number of phages are already occupied by non-specific adsorption prior to positive selection. ...
Pancreatic cancer is a devastating disease with a high mortality rate and a lack of effective therapies. The challenges associated with early detection and the highly aggressive nature of pancreatic cancer have limited treatment options, underscoring the urgent need for better disease-modifying therapies. Peptide-based biotherapeutics have become an attractive area of research due to their favorable properties such as high selectivity and affinity, chemical modifiability, good tissue permeability, and easy metabolism and excretion. Phage display, a powerful technique for identifying peptides with high affinity and specificity for their target molecules, has emerged as a key tool in the discovery of peptide-based drugs. Phage display technology involves the use of bacteriophages to express peptide libraries, which are then screened against a target of interest to identify peptides with desired properties. This approach has shown great promise in cancer diagnosis and treatment, with potential applications in targeting cancer cells and developing new therapies. In this comprehensive review, we provide an overview of the basic biology of phage vectors, the principles of phage library construction, and various methods for binding affinity assessment. We then describe the applications of phage display in pancreatic cancer therapy, targeted drug delivery, and early detection. Despite its promising potential, there are still challenges to be addressed, such as optimizing the selection process and improving the pharmacokinetic properties of phage-based drugs. Nevertheless, phage display represents a promising approach for the development of novel targeted therapies in pancreatic cancer and other tumors.
... In order to avoid them, "subtractive biopanning" is advisable-that is, incubating the initial library onto the biopanning infrastructure containing all elements except the target to eliminate all potential TUPs. The most comprehensive list to date of TUP amino acid sequences reported in the literature is the "Scanner And Reporter Of Target-Unrelated Peptides" (SAROTUP) [93,94]. Subtractive biopanning may also be used to find a peptide with higher selectivity. ...
Foodborne pathogens present a serious issue around the world due to the remarkably high number of illnesses they cause every year. In an effort to narrow the gap between monitoring needs and currently implemented classical detection methodologies, the last decades have seen an increased development of highly accurate and reliable biosensors. Peptides as recognition biomolecules have been explored to develop biosensors that combine simple sample preparation and enhanced detection of bacterial pathogens in food. This review first focuses on the selection strategies for the design and screening of sensitive peptide bioreceptors, such as the isolation of natural antimicrobial peptides (AMPs) from living organisms, the screening of peptides by phage display and the use of in silico tools. Subsequently, an overview on the state-of-the-art techniques in the development of peptide-based biosensors for foodborne pathogen detection based on various transduction systems was given. Additionally, limitations in classical detection strategies have led to the development of innovative approaches for food monitoring, such as electronic noses, as promising alternatives. The use of peptide receptors in electronic noses is a growing field and the recent advances of such systems for foodborne pathogen detection are presented. All these biosensors and electronic noses are promising alternatives for the pathogen detection with high sensitivity, low cost and rapid response, and some of them are potential portable devices for on-site analyses.
... Phage display has been used for performing high-throughput screening and identifying functional peptide ligands for various targets, including PD-L1 [28], tumor stromal cells [29], and ischemic myocardial tissues [30]. These ligands offer targeting fragments for the construction of efficient diagnostic and therapeutic platforms [31]. In this study, we identified Content courtesy of Springer Nature, terms of use apply. ...
Background
Effective therapeutics to stop or reverse liver fibrosis have not emerged, because these potential agents cannot specifically target activated hepatic stellate cells (aHSCs) or are frequently toxic to parenchymal cells. Human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes show promise in nanomedicine for the treatment of liver fibrosis. However, systemic injection showed that unmodified exosomes were mainly taken up by the mononuclear phagocyte system. The discovery of ligands that selectively bind to a specific target plays a crucial role in clinically relevant diagnostics and therapeutics. Herein, we aimed to identify the targeting peptide of aHSCs by screening a phage-displayed peptide library, and modify Huc-MSC-derived exosomes with the targeting peptide.
Results
In this study, we screened a phage-displayed peptide library by biopanning for peptides preferentially bound to HSC-T6 cells. The identified peptide, HSTP1, also exhibited better targeting ability to aHSCs in pathological sections of fibrotic liver tissues. Then, HSTP1 was fused with exosomal enriched membrane protein (Lamp2b) and was displayed on the surface of exosomes through genetic engineering technology. The engineered exosomes (HSTP1-Exos) could be more efficiently internalized by HSC-T6 cells and outperformed both unmodified exosomes (Blank-Exos) and Lamp2b protein overexpressed exosomes (Lamp2b + Exos) in enhancing the ability of exosomes to promote HSC-T6 reversion to a quiescent phenotype. In vivo results showed HSTP1-Exos could specifically target to the aHSC region after intravenous administration, as demonstrated by coimmunofluorescence with the typical aHSCs marker α-SMA, and enhance the therapeutic effect on liver fibrosis.
Conclusion
These results suggest that HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs and that HSTP1-modified exosomes realize the precise treatment for aHSCs in complex liver tissue. We provide a novel strategy for clinical liver fibrosis therapy.
Graphical Abstract
... This analysis is limited to hundreds of clones, and it often results in repetitive isolation of the same clones that become dominant during selection cycles. In addition, clone enrichment can also reflect features not necessarily related to target affinity, including biological fitness and binding to non-target substrates (discussed in the next sections) reducing the isolation of true binders [52]. ...
... Obviously, this issue also disturbs the screening of phage display peptide libraries. The Huang's and Smith's labs have developed bioinformatic tools, mainly based on the target-unrelated peptides data bank (predicted and verified) to discard a priori these clones streamlining the peptide discovery process [52,[81][82][83][84][85][86]. By collecting growing NGS data, we are developing a similar tool for scFv phage display libraries, described in the next sections, that would be of potential interest for the whole scientific community involved in antibody discovery. ...
Simple Summary
Monoclonal antibodies are increasingly used for a broad range of diseases. Rising demand must face with time time-consuming and laborious processes to isolate novel monoclonal antibodies. Next-generation sequencing coupled to phage display provides timely and sustainable high throughput selection strategy to rapidly access novel target. Here, we describe the current NGS-guided strategies to identify potential binders from enriched sub-libraires by applying a user-friendly informatic pipeline to identify and discard false positive clones. Rescue step and strategies to boost mAb yield are also discussed to improve the limiting selection and screening steps.
Abstract
Monoclonal antibodies are among the most powerful therapeutics in modern medicine. Since the approval of the first therapeutic antibody in 1986, monoclonal antibodies keep holding great expectations for application in a range of clinical indications, highlighting the need to provide timely and sustainable access to powerful screening options. However, their application in the past has been limited by time-consuming and expensive steps of discovery and production. The screening of antibody repertoires is a laborious step; however, the implementation of next-generation sequencing-guided screening of single-chain antibody fragments has now largely overcome this issue. This review provides a detailed overview of the current strategies for the identification of monoclonal antibodies from phage display-based libraries. We also discuss the challenges and the possible solutions to improve the limiting selection and screening steps, in order to keep pace with the increasing demand for monoclonal antibodies.
... The essence of phage display lies in the physical presentation of peptides on the bacteriophage surface coupled with a selection strategy for rapid affinity isolation of binders (Bakhshinejad et al., 2016;Valldorf et al., 2021). The selection strategy allows researchers to define order from the massive chaos conferred by the random library (Castel et al., 2011;Gray and Brown, 2014;Molek et al., 2011). ...
The immune system is tasked to keep our body unharmed and healthy. In the immune system, B- and T-lymphocytes are the two main components working together to stop and eliminate invading threats like virus particles, bacteria, fungi and parasite from attacking our healthy cells. The function of antibodies is relatively more direct in target recognition as compared to T-cell receptors (TCR) which recognizes antigenic peptides being presented on the major histocompatibility complex (MHC). Although phage display has been widely applied for antibody presentation, this is the opposite in the case of TCR. The cell surface TCR is a relatively large and complex molecule, making presentation on phage surfaces challenging. Even so, recombinant versions and modifications have been introduced to allow the growing development of TCR in phage display. In addition, the increasing application of TCR for immunotherapy has made it an important binding motif to be developed by phage display. This review will emphasize on the application of phage display for TCR discovery as well as the engineering aspect of TCR for improved characteristics.
... In 1985, George Smith first described phage display by demonstrating the ability of filamentous phage to display peptide by fusing a peptide sequence library to a viral capsid protein (Smith 1985). Phage display technology has been used and promoted for the identification of targeting ligands that specifically bind to a variety of targets and has become a mature technology (Bakhshinejad et al. 2016). Tang (2013) developed a panning procedure using a phage display peptide library to select a peptide that specifically binds to the VPAC1 receptor to develop a novel targeted probe for molecular imaging and therapy. ...
Treatments of brain diseases are heavily limited by the existence of the blood–brain barrier (BBB), which precludes efficient drug delivery to the brain. Compared with the BBB, drugs may have a better likelihood of reaching the brain via the cerebrospinal fluid (CSF) because of the lack of a barrier between the CSF and the brain. In this study, phage display technology was effectively applied to screen novel peptides as targeting motifs to transport drugs across the blood-cerebrospinal fluid barrier (BCSFB). We applied a phage seven-mer cyclic peptide library (Ph.D.-C7C™) intravenously to rats and later recovered phages from the CSF. After several rounds of screening, the candidate phages that could cross the BCSFB were enriched. Several bacteriophage clones from the final round were randomly selected and sequenced. A peptide sequence denoted as PMK, which was demonstrated to be able to cross the BCSFB via in vivo optical imaging analysis, could be used in the future for the construction of targeted drug delivery systems.
