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Schematic description of synchronous in vivo phage display screening. In every selection round, phage are intravenously (i.v.) administered and simultaneously recovered from N target tissues, amplified, pooled, and used for the next selection round. Increased recovery of phage transforming units (TU) in the third round reflects the selection of peptides preferentially homing to the target organ.
Source publication
In vivo phage display is a technology used to reveal organ-specific vascular ligand-receptor systems in animal models and, recently, in patients, and to validate them as potential therapy targets. Here, we devised an efficient approach to simultaneously screen phage display libraries for peptides homing to any number of tissues without the need for...
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The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of speci...
Citations
... Kolonin и соавт. (2006) выполнили скрининг с комбинаторной библиотекой пептидов у мышей и охарактеризовали пептид, который может избирательно связываться с сосудистым эндотелием БЖТ [55]. Визуализация in vivo с использованием пептидной пробы 3, меченной инфракрасной флуоресцирующей меткой IRDye800 (PEP3-IRDye800), показала накопление в БЖТ, сигнал увеличивался после обработки холодом. ...
Epicardial adipose tissue (EAT) is a visceral depot of the heart fat, which has high plasticity and directly contact with the myocardium and coronary arteries. Epicardial fat is a unique paracrine organ closely anatomically and physiologically related to the myocardium. Recent studies have repeatedly confirmed the role of epicardial fat in the progression of the cardiovascular diseases. The accumulation of EAT, measured by using new non-invasive imaging techniques, is prospectively associated with the onset and progression of coronary heart disease (CHD) and atrial fibrillation. This review focuses on modern in vivo methods for assessing epicardial fat.
... Phage-displayed peptide screenings performed in vivo or on cell cultures face the plethora of in vivo expressed target molecules and, consequently, generate the recovery of complex repertoires of binding peptides. Next-generation sequencing (NGS) technologies have shown the high complexity of isolated peptide repertoires [15]. 2 of 13 Despite the development of software solutions for the NGS analysis of the comparable complexity of generated peptide repertoires [16] the limited choice of targeting peptides was mainly caused by either the enrichment of the main consensus peptide sequences during the selection procedures, or the particular presence of a number of peptides when compared to control selections. This is further supported by physical subtraction experiments of comparable target vs. control selected phage repertoires [8]. ...
Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections.
... To identify crotamine-binding peptides, we employed a phage display library screening approach routinely used by our group [22,23,[26][27][28][29][30][31]. Four rounds of library biopanning on crotamine were performed to enrich for crotaminebinding phage-displayed peptides. ...
Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine.
Key messages
Synthetic crotamine was characterized as a functional substitute for venom-derived crotamine based on myotoxic effects.
A combinatorial peptide library was screened for crotamine-binding peptides.
Tryptophan-rich peptides were shown to bind to crotamine and interfere with its DNA binding.
Crotamine myofiber distribution and affinity for tryptophan-rich peptides provide insights on its mechanism of action.
... Simultaneous identification of multiple pairs of targeted organs/peptides has also been accomplished using the same in vivo phage display approach. [8][9][10] Such in vivo panning has been performed both in mice and in humans. 11 Complementary in vivo panning can start from injection of phage in circulation and identify phage clones that do not home to any organs. ...
... A random peptide library may display tens of millions of peptide epitope, which makes phage display derived products play a significant role in the diagnosis and treatment of diseases. Macromolecules, bacteria, cells, tissues, organs, animals, and even nanoparticles [50][51][52][53][54][55] can all serve as the targets for bio-panning to obtain specific binding peptides. Therefore, many peptide mimotopes of mycotoxins have been identified through affinity selection from phage display libraries (Table 1), such as OTA, AFB 1 , FB 1 and DON et al. ...
Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.
... Simultaneous identification of multiple pairs of targeted organs/peptides has also been accomplished using the same in vivo phage display approach. [8][9][10] Such in vivo panning has been performed both in mice and in humans. 11 Complementary in vivo panning can start from injection of phage in circulation and identify phage clones that do not home to any organs. ...
Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo . However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA 5 -phage) and 50 copies of ScA on p8 protein (ScA 50 -phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo . A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo . In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo .
Abstract Graphic
... Peptides containing the SRL motif showed uptake in the brain several folds higher than that observed in the kidney. In 2006, Kolonin and coworkers, using the same approach, identified another tripeptide motif targeting brain vasculature, LGG (Leu-Gly-Gly) [94]. Rooy and coworkers, in 2010, used phage display in an in situ perfusion model for the screening of new brain endothelium peptide ligands. ...
The main obstacle to biopharmaceutical delivery in therapeutic concentration into the brain for treating neurological disorders is the presence of the blood-brain barrier (BBB). The physiological process of receptor-mediated transcytosis (RMT) to transport cargo through the brain endothelial cells toward brain parenchyma has prompted researchers to search for non-natural ligands that can be used to transport drugs across the BBB. Conjugation of drugs to RMT ligands would be an effective strategy for its delivery to the central nervous system. An attractive approach to identify novel transcytosing ligands is the screening by phage display combinatorial libraries. The main technology strength lies in the large variety of exogenous peptides or proteins displayed on the phage's surface. Here, we provide a mini-review of phage display technology using in vitro and in vivo BBB models for the development of peptide-mediated drug delivery systems.
... The development of in vivo phage-display technology has enabled researchers to isolate phage particles selectively targeting the vasculature of individual internal organs [31]. In addition, hybrid prokaryotic-eukaryotic vectors capable of transducing mammalian cells through ligand-directed targeting to a specific receptor were reported; these vectors, termed AAVP, are chimeras of recombinant adeno-associated viruses and phage [32]. ...
Introduction: Bacteriophages are an abundant component of the mucosal microbiota in humans and some
animal species. Intestinal epithelial cells (IECs) are the key element responsible for the induction
and regulation of immune responses in the gut mucosa. The objective of this study was
to evaluate the effects of T4 and A5/80 bacteriophages on the expression of immunologically
important genes in Caco-2, a model cell line for IECs.
Materials & Method: Bacteriophages were added to cultures of differentiated Caco-2 cells for 12 hours, while
control cultures were treated with phosphate-buffered saline (PBS). Expression of genes in
Caco-2 cells was determined using custom-made RT2 Profiler PCR Arrays, which allow for
the evaluation of gene expression with the sensitivity and specificity of real-time PCR. We
evaluated the expression of 21 genes which are important for the immune functions of IECs,
including IL1B, IL6, IL7, IL10, IL15, IL18, IL25, IL33, TGFB1, TNF, CXCL8, CCL2, TSLP, FCER2, PIGR, DEFB4A,
CAMP, REG3G, TNFSF13, TNFSF13B, and MUC2.
Results: Both examined phages significantly influenced the expression of a number of genes compared
with control cultures. In particular, T4 significantly increased the expression of the CCL2 and
DEFB4A genes, while A5/80 induced the expression of the PIGR gene.
Discussion: Together with the findings from previous studies, our results suggest that by modulating the
expression of some genes, bacteriophages may affect immune responses in the gut mucosa.
... Previous attempts to identify a brain delivery vehicle using in vivo phage display technology targeted the cerebral vasculature (26)(27)(28)(29), the CSF barrier (30,31) or the olfactory tract (32) but not the brain parenchyma. Moreover, the transport mechanism for all of these targeting peptides and antibodies remains unknown. ...
Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high-affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB.
Methods
A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. Phage formatted VNARs cross-reactive to recombinant human and mouse TfR1-ECD were fused to Fc domain of human IgG1 (hFc) and tested for TfR1-ECD binding by ELISA and surface plasmon resonance. The pharmacokinetics and biodistribution of VNAR-hFcs were studied in mice by ELISA and immunolabeling following intravenous (IV) injection and cardiac perfusion. Functional activity was measured by body temperature reduction following the IV injection of neurotensin fused to a TXB2-hFc (TXB2-hFc-NT).
Results
TXB2 was identified as a high-affinity, species cross-reactive VNAR antibody against TfR1-ECD, that does not to compete with transferrin or ferritin for receptor binding. IV dosing of TXB2-hFc at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc-NT at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung.
Conclusions
A species cross-reactive and brain-selective VNAR antibody to TfR1 was identified by a combination of in vitro and in vivo phage selection. As a high-affinity, bivalent Fc fusion protein, TXB2 rapidly crossed the BBB and exhibited a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.
... To identify tissue-selective 3-mer motifs observed in each tissue, we performed a Fisher's exact test on each motif, as suggested previously by [52]. For each identified motif recovered per tissue, a 2 × 2 contingency table comparing the frequency of each motif observed in the isolated tissue with the frequency of each motif observed in the normalizing sample was prepared and a p-value calculated using a one-sided Fisher's exact test, implemented in a custom script using the Fisher exact function from the scipy (v. ...
... Given the Viruses 2019, 11, 988 13 of 21 lack of tissue-specific motif enrichment, we speculate that most phages in the liver and spleen were accumulated via motif-independent processes that were largely independent upon the peptide structure displayed on p8. From these data, we demonstrate that 3-mer motifs were enriched in a tissue-specific manner at the earliest stages of in vivo evolution, as confirmed by previous studies [33,52,69]. ...
... Given the lack of tissue-specific motif enrichment, we speculate that most phages in the liver and spleen were accumulated via motif-independent processes that were largely independent upon the peptide structure displayed on p8. From these data, we demonstrate that 3-mer motifs were enriched in a tissue-specific manner at the earliest stages of in vivo evolution, as confirmed by previous studies [33,52,69]. Figure 5. Identification of tissue-selective motifs by tissue. ...
Peptide-displayed phage libraries are billion-clone collections of diverse chimeric bacteriophage particles, decorated by genetically fused peptides built from a random combination of natural amino acids. Studying the molecular evolution of peptide-displayed libraries in mammalian model systems, using in vivo phage display techniques, can provide invaluable knowledge about the underlying physiology of the vasculature system, allow recognition of organ- and tissue-specific networks of protein–protein interactions, and provide ligands for targeted diagnostics and therapeutics. Recently, we discovered that landscape phage libraries, a specific type of multivalent peptide phage display library, expose on their surface comprehensive collections of elementary binding units (EBUs), which can form short linear motifs (SLiMs) that interact with functional domains of physiologically relevant proteins. Because of their unique structural and functional features, landscape phages can use an alternative mechanism of directed molecular evolution, i.e., combinatorial avidity selection. These discoveries fueled our interest in revisiting the in vivo evolution of phage displayed libraries using another format of display, i.e., landscape phages. In this study, we monitored the evolution of a landscape phage library in a mouse model with and without an implanted human breast cancer tumor xenograft. As expected, the multivalent architecture of landscape phage displayed proteins provided strong tissue selectivity and resulted in a huge diversity of tissue penetrating, chimeric phage particles. We identified several types of EBU interactions that evolved during the course of tissue distribution, which included interactions of EBUs with all tissue types, those EBUs that interacted selectively with specific organs or tissues with shared gene expression profiles or functionalities, and other EBUs that interacted in a tissue-selective manner. We demonstrated that landscape phage libraries are a rich collection of unique nanobioparticles that can be used to identify functional organ and tissue-binding elements after the evolution of a phage display library in vivo.
