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In vivo phage-display screening for peptides that home to human tissues through the systemic circulation. a, Schematic flowchart of the study. b, Phage recovery from various human tissues in vivo. Tissue samples were processed and phage recovered as described in Methods. Shown are means s.e.m. of phage transducing units (TU) per gram of tissue obtained from each biopsy site.
Source publication
The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized...
Contexts in source publication
Context 1
... patient (see Methods) received an intravenous infusion of the unselected random phage library, and 15 min after infu- sion tissue biopsies were obtained to provide histopathological diagnosis and to recover phage from various organs (Fig. ...
Citations
... Multiple research groups have administered phage libraries (up to 10 14 phage particles) to patients and conducted in vivo bio-panning to identify tissuespecific peptides without noticeable side effects. This highlights their excellent tolerance in humans (Arap et al., 2002;Krag et al., 2006;Shukla et al., 2013;Christiansen et al., 2015). 5) Phages possess the unique ability to form highly ordered structures such as liquid crystals at certain concentrations (Sawada, 2017). ...
Bacteriophages, also known as phages, are viruses that replicate in bacteria and archaea. Phages were initially discovered as antimicrobial agents, and they have been used as therapeutic agents for bacterial infection in a process known as “phage therapy.” Recently, phages have been investigated as functional nanomaterials in a variety of areas, as they can function not only as therapeutic agents but also as biosensors and tissue regenerative materials. Phages are nontoxic to humans, and they possess self-assembled nanostructures and functional properties. Additionally, phages can be easily genetically modified to display specific peptides or to screen for functional peptides via phage display. Here, we demonstrated the application of phage nanomaterials in the context of tissue engineering, sensing, and probing.
... Normal organs and diseased sites express unique molecular markers such as endothelial cell surface receptors that are accessible from the circulation and enable tissue-specific targeting by ligands upon systemic administration (1)(2)(3)(4)(5)(6)(7)(8)(9). Over the past ~25 y, these ligands, along with their corresponding receptors, have been identified on the vascular endothelium of normal organs (1)(2)(3)(4) and tumors (5)(6)(7)(8)(9)(10) in experimental animal models (1)(2)(3)(4)(5)(6) and even directly in patients (4,(7)(8)(9). ...
... Normal organs and diseased sites express unique molecular markers such as endothelial cell surface receptors that are accessible from the circulation and enable tissue-specific targeting by ligands upon systemic administration (1)(2)(3)(4)(5)(6)(7)(8)(9). Over the past ~25 y, these ligands, along with their corresponding receptors, have been identified on the vascular endothelium of normal organs (1)(2)(3)(4) and tumors (5)(6)(7)(8)(9)(10) in experimental animal models (1)(2)(3)(4)(5)(6) and even directly in patients (4,(7)(8)(9). This vascular diversity has resulted in applications that include targeted drug delivery, molecular imaging, and disease modeling (3)(4)(5)(6)(8)(9)(10)(11). ...
... Normal organs and diseased sites express unique molecular markers such as endothelial cell surface receptors that are accessible from the circulation and enable tissue-specific targeting by ligands upon systemic administration (1)(2)(3)(4)(5)(6)(7)(8)(9). Over the past ~25 y, these ligands, along with their corresponding receptors, have been identified on the vascular endothelium of normal organs (1)(2)(3)(4) and tumors (5)(6)(7)(8)(9)(10) in experimental animal models (1)(2)(3)(4)(5)(6) and even directly in patients (4,(7)(8)(9). This vascular diversity has resulted in applications that include targeted drug delivery, molecular imaging, and disease modeling (3)(4)(5)(6)(8)(9)(10)(11). ...
The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.
... Many different types of viruses, both mammalian and bacterial, have been used for several types of biological applications [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Bacteriophages, i.e., prokaryotic viruses that exclusively infect bacteria, not mammalian cells, have recently attracted the attention of the scientific community as potential cancer immunotherapy agents. ...
... Biopanning is an effective selection technique that enriches a diverse phage library by immobilizing it on a receptor on a solid support, washing away unbound phages, and resulting in an enriched phage population with high affinity for the specific receptor [85]. This technique is used to isolate peptides that bind to a specific target molecule based on their binding affinities [10,85,86]. ...
... Phages are also effective in targeting specific tissues and organs for drug delivery or imaging, including the brain, retina, skin, pancreas, lungs, adrenal glands, uterus, and intestines [10,90,94]. Targeting specific tissues and organs can be challenging because of the relative impermeability of the blood-brain barrier (BBB). ...
Bacteriophages have emerged as versatile tools in the field of bioengineering, with enormous potential in tissue engineering, vaccine development, and immunotherapy. The genetic makeup of phages can be harnessed for the development of novel DNA vaccines and antigen display systems, as they can provide a highly organized and repetitive presentation of antigens to immune cells. Bacteriophages have opened new possibilities for the targeting of specific molecular determinants of cancer cells. Phages can be used as anticancer agents and carriers of imaging molecules and therapeutics. In this review, we explored the role of bacteriophages and bacteriophage engineering in targeted cancer therapy. The question of how the engineered bacteriophages can interact with the biological and immunological systems is emphasized to comprehend the underlying mechanism of phage use in cancer immunotherapy. The effectiveness of phage display technology in identifying high-affinity ligands for substrates, such as cancer cells and tumor-associated molecules, and the emerging field of phage engineering and its potential in the development of effective cancer treatments are discussed. We also highlight phage usage in clinical trials as well as the related patents. This review provides a new insight into engineered phage-based cancer vaccines.
... In this technique, the phage displays the expressed peptides on its surface, followed by the development of a specific peptide that binds strongly to a particular target [19][20][21][22]. This technique has been widely used in the study of selecting functional peptides that bind to a target, because it has the advantage of amplifying phages with the desired peptide and analyzing the sequence to identify the amino acids of peptides [23,24]. For example, Cho, et al. discovered affinity peptides that bind specifically to interleukin-33 (IL-33) using the phage display technique and used the specific peptide to achieve drug development or allergy diagnosis [25]. ...
Herein, we have developed peptide-coated gold nanoparticles (AuNPs) based on localized surface plasmon resonance (LSPR) sensor chips that can detect fipronil with high sensitivity and selectivity. The phage display technique has been exploited for the screening of highly specific fipronil-binding peptides for the selective detection of the molecule. LSPR sensor chips are fabricated initially by attaching uniformly synthesized AuNPs on the glass substrate, followed by the addition of screened peptides. The parameters, such as the peptide concentration of 20 µg mL−1 and the reaction time of 30 min, are further optimized to maximize the efficacy of the fabricated LSPR sensor chips. The sensing analysis is performed systematically under standard fipronil solutions and spike samples from eggs. The developed sensor has shown excellent sensitivity towards both standard solutions and spike samples with limit of detection (LOD) values of 0.01 ppb, respectively. Significantly, the developed LSPR sensor chips offer distinct features, such as a facile fabrication approach, on-site sensing, rapid analysis, cost-effectiveness, and the possibility of mass production, in which the chips can be effectively used as a promising and potential on-site detection tool for the estimation of fipronil.
... Since then, in vivo use of phage display has allowed the identification of several receptor-ligand pairs in malignant and benign disease contexts and different animal models. Interestingly, in 2002 the first in vivo phage display in humans was performed by Arap et al. (2002b). In this work an in vivo screening of a phage displayed peptide library was performed to investigate the molecular diversity of receptors specific of human vascular beds. ...
... This large-scale screening indicated that the tissue distribution of circulating peptides was nonrandom. To validate the specificity of the isolated peptides, the interaction of an IL-11 peptide mimic isolated from the phage display screening to interleukin-11 receptor (IL-11R) of normal prostate endothelium and epithelium tissue was confirmed (Arap et al., 2002b). Later, IL-11R was validated by Zurita et al. (2004) as a molecular target for prostate cancer therapeutics. ...
... Later, IL-11R was validated by Zurita et al. (2004) as a molecular target for prostate cancer therapeutics. Afterward, the library obtained by Arap et al. (2002b) was further screened in two subsequent cancer patients to uncover ligand-receptors common or specific to certain vascular beds. These results allowed the identification of four native ligand-receptors, three of which have not been previously reported. ...
The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity responses have led to the development of alternative approaches for the streamlined identification of most effective antibodies. Within this context, display selection technologies such as phage display, ribosome display, yeast display, bacterial display, and mammalian cell surface display have been widely promoted over the past three decades as ideal alternatives to traditional hybridoma methods. The display of antibodies on phages is probably the most widespread and powerful of these methods and, since its invention in late 1980s, significant technological advancements in the design, construction, and selection of antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. With evolving novel disease targets and the emerging of a new generation of therapeutic antibodies, such as bispecific antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapies, it is clear that phage display is expected to continue to play a central role in antibody development. Nevertheless, for non-standard and more demanding cases aiming to generate best-in-class therapeutic antibodies against challenging targets and unmet medical needs, in vivo phage display selections by which phage libraries are directly injected into animals or humans for isolating and identifying the phages bound to specific tissues offer an advantage over conventional in vitro phage display screening procedures. Thus, in the present review, we will first summarize a general overview of the antibody therapeutic market, the different types of antibody fragments, and novel engineered variants that have already been explored. Then, we will discuss the state-of-the-art of in vivo phage display methodologies as a promising emerging selection strategy for improvement antibody targeting and drug delivery properties.
... Our data are consistent with the phenotype observed upon brown adipocyte ablation via a transgenic toxigene approach [13]. Our hunter killer-peptide design had been previously used for other receptor-binding peptides and taken to the clinic [64,69]. A peptide targeting WAT has been shown to reverse obesity and increase metabolism in several animal models [30,68]. ...
Objective
Brown adipogenesis and thermogenesis in brown and beige adipose tissue (AT) involve vascular remodeling and sympathetic neuronal guidance. Here, we investigated the molecular mechanism coordinating these processes.
Methods
We used mouse models to identify the molecular target of a peptide CPATAERPC homing to the endothelium of brown and beige AT.
Results
We demonstrate that CPATAERPC mimics nerve growth factor (NGF) and identify a low molecular weight isoform of NGF receptor, TrkA, as the CPATAERPC cell surface target. We show that the expression of truncated endothelial TrkA is selective for brown and subcutaneous AT. Analysis of mice with endothelium-specific TrkA knockout revealed the role of TrkA in neuro-vascular coordination supporting the thermogenic function of brown adipocytes. A hunter-killer peptide D-BAT, composed of CPATAERPC and a pro-apoptotic domain, induced cell death in the endothelium and adipocytes. This resulted in thermogenesis impairment, and predisposed mice to obesity and glucose intolerance. We also tested if this treatment can inhibit the tumor recruitment of lipids mobilized from adipocytes from adjacent AT. Indeed, in a mouse model of breast cancer D-BAT suppressed tumor-associated AT lipolysis, which resulted in reduced fatty acid utilization by cancer cells.
Conclusion
Our study demonstrates that TrkA signaling in the endothelium supports neuro-vascular coordination enabling beige adipogenesis.
... Phages are abundant, simplistic viruses that have no native tropism for mammalian tissues, are not pathogenic, and are economical and efficient to manipulate and produce at GMP standards (Regulski et al, 2021). The use of phages as an antibiotic was widely accepted and used in the pre-antibiotic era, but in modern laboratory research, they play an important role in drug discovery in vitro and in vivo by their ability to tolerate large mutations on their coat proteins with very high binding specificity (Pasqualini & Ruoslahti, 1996;Arap et al, 2002;Kutateladze & Adamia, 2010;Bradbury et al, 2011). As a result, the use of phages in mammalian gene delivery has been explored through the insertion of a mammalian or viral transgene cassettes in its genome, and a receptor-specific mutation on its coat protein genes (Larocca et al, 1998(Larocca et al, , 1999(Larocca et al, , 2001Burg et al, 2002). ...
Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNF α ) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses.
... Under a homogeneous in situ process, all 96 wells were coated with a single specific target, which enabled affirmation over zero hindrance in targetspecific isolation. Yet, for a better assurance on the application with an animal model, more species-specific and live advanced experimentation is required, for which in vivo and in vitro techniques are employed [53]. With other precise approaches, the ability to screen undefined cell surface receptors internalized peptides and experience a high-throughput screening of single cell-binding multiple peptides, in vitro cell screening technology is done [54,55]. ...
... The works on murine models a significant reduction in tumor growth rate. Other prominent applications include exploiting the technology to isolate peptides predominantly expressed in tumor blood vessels [53]. ...
Introduction:
There is a constant drive to improve disease treatments. Much effort has been directed at identifying less immunogenic anti-cancer agents that produce fewer and less severe side effects. For more than a decade, bacteriophages have been discussed as an effective treatment for cancer with an exact mode of delivery.
Areas covered:
We review how bacteriophages are used in cancer treatment, the underlying therapeutic mechanisms, and the tumour attacking peptide screening process. The filamentous bacteriophages are an effective vehicle for delivering displayed peptides toward the tumour target. The peptide must be expressed at the appropriate coat protein, and the peptide must be effective enough to disrupt the complex cancer matrix. The present review also sheds light on the dynamic use of phage in cancer treatment, from detection and diagnostics to treatment.
Expert opinion:
Phage has a versatile role as a diagnostic and therapeutic tool. By acting as an appropriate recombinant drug, this phage has every potential to replace existing laborious, high capital investing therapies that may at many times result in failure or drastic side effects. One of the most significant challenges would be identifying tumour homing peptides. Although a few have been discovered, the most effective ones are yet to be determined. This therapeutic method plays a significant role in tumour therapy with high accuracy and efficiency, irrespective of the target location.
... While these methods are often used to study intracellular protein levels, several groups have focused their attention on the luminal surface of the endothelium, which is in immediate contact with the blood. [50][51][52] Not surprisingly, these studies also identified (interactive regions of) proteins being present in organ-specific patterns, and this knowledge could potentially be used to develop methods and therapeutics that target specific vascular beds. ...
A State of the Art lecture entitled “Molecular Analysis of Vascular Gene Expression” was presented at the ISTH Congress in 2021. Endothelial cells (ECs) form a critical interface between the blood and underlying tissue environment, serving as a reactive barrier to maintain tissue homeostasis. ECs play an important role in not only coagulation, but also in the response to inflammation by connecting these two processes in the host defense against pathogens. Furthermore, ECs tailor their behavior to the needs of the microenvironment in which they reside, resulting in a broad display of EC phenotypes. While this heterogeneity has been acknowledged for decades, the contributing molecular mechanisms have only recently started to emerge due to technological advances. These include high‐throughput sequencing combined with methods to isolate ECs directly from their native tissue environment, as well as sequencing samples at a high cellular resolution. In addition, the newest technologies simultaneously quantitate and visualize a multitude of RNA transcripts directly in tissue sections, thus providing spatial information. Understanding how ECs function in (patho)physiological conditions is crucial to develop new therapeutics as many diseases can directly affect the endothelium. Of particular relevance for thrombotic disorders, EC dysfunction can lead to a procoagulant, proinflammatory phenotype with increased vascular permeability that can result in coagulopathy and tissue damage, as seen in a number of infectious diseases, including sepsis and coronavirus disease 2019. In light of the current pandemic, we will summarize relevant new data on the latter topic presented during the 2021 ISTH Congress.
... In the past two decades, it has been reported that TECs exhibited some abnormal phenotypes compared with NECs. 5 For instance, NEC and TEC gene expression patterns are different. [6][7][8][9][10][11] TECs proliferate and migrate faster than NECs. 12,13 Several upregulated genes in TECs are associated with angiogenesis. ...
... Several research groups, including ours, have found that some genes are upregulated in TECs compared with NECs. [6][7][8][9][10][11] Those molecules can be candidates for TEC-targeting therapy. Specifically, another strategy is delivering a drug into tumor blood vessels using the nano DDS system. ...
Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner, leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as the multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) into MEND because αvβ3 integrin, a receptor of cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized by biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.
