Screening Of Phage-displayed Peptide Libraries Research Articles

A Novel CTLA-4 affinity peptide for cancer immunotherapy by increasing the integrin αvβ3 targeting

Immune checkpoint inhibitors (ICIs) are changing all aspects of malignant tumour therapy as an immunotherapy subverter in oncology. However, the current ICIs might induce systemic immune activation in other tissues and organs since they are not tumour-specific, causing the immune system to attack some normal tissues and organs of the human body. The toxicity can also amplify greatly although combined immunotherapy for cancer has increased the curative efficacy. The LC4 peptide was modified to improve its tumour-targeting ability and reduce peripheral immune system activation, which was obtained through phage display peptide library screening and could block the CTLA-4/CD80 interaction. The LC4 peptide as a result, like other ICIs, exerts anti-tumour effects by refreshing T cell function, and also activates the peripheral immune system. We used the PLGLAG peptide as a linker at the C-terminal of LC4 to connect with a tumour-targeting peptide RGD to increase the tumour tissue targeting ability, and obtain LC4-PLG-RGD. Further experiments demonstrated that the anti-tumour LC4-PLG-RGD activity was better than LC4 in vivo, and the ability to activate the peripheral immune system was weakened. In conclusion, LC4-PLG-RGD can increase the ICIs tumour-targeting and reduce excessive peripheral tissue immune activation, thereby reducing the side effects of ICIs, while increasing their anti-tumour efficacy. This study confirmed that enhanced ICI tumour targeting can effectively reduce immune-related adverse reaction occurrence.

Development of a novel hybrid antimicrobial peptide for targeted killing of Pseudomonas aeruginosa

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa, coupled with shrinking antibiotic pipelines, has increased the demand for new antimicrobials with novel mechanisms of action. As the indiscriminate nature of broad-spectrum antimicrobial toxicity may have negative clinical consequences and increase the incidence of resistance, we have developed a P. aeruginosa-selective antimicrobial peptide capable of preferentially killing P. aeruginosa relative to benign microorganisms. A targeting peptide (PA2) that binds specifically to OprF porin on P. aeruginosa was identified by phage display peptide library screening, and a hybrid peptide was constructed by addition of the targeting peptide to GNU7, a potent antimicrobial peptide. The resulting hybrid peptide PA2-GNU7 exhibited potent antimicrobial activity against P. aeruginosa without causing host toxicity. Confocal laser scanning microscopy analysis and time-kill experiments demonstrated that PA2-GNU7 exhibited a high degree of specificity for P. aeruginosa, and rapidly and selectively killed P. aeruginosa cells in mixed cultures. In addition, in vivo treatment efficacy of PA2-GNU7 was significantly greater than that of conventional antibiotics in a mouse model of MDR P. aeruginosa infection. Taken together, the data suggest that PA2-GNU7 may be a promising template for further development as a novel anti-MDR P. aeruginosa therapeutic agent.

Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.

Disintegrin-like domain of glycoprotein B regulates Kaposi's sarcoma-associated herpesvirus infection of cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein's interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.

Hepatitis A virus mimotope mapping by phage display peptide library

A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach. Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. 10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV. The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.

Discovery and Structural Characterization of a Phospholamban‐Binding Cyclic Peptide and Design of Novel Inhibitors of Phospholamban

The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.

Identification of a Novel Skin Penetration Enhancement Peptide by Phage Display Peptide Library Screening

Skin is an important site for local or systemic application of drugs. However, a majority of drugs have poor permeability through the skin's topmost layer, stratum corneum (SC). The aim of this study was to identify safe and smaller peptides that could enhance the skin penetration of drug molecules. By screening phage display peptide library, we have identified a T2 peptide (LVGVFH), which enhanced the penetration of bacteriophages (~800 nm long bacterial viruses) across porcine and mouse skin. Pretreating the skin with synthetic T2 peptide at pH 4.5 resulted in significant penetration enhancement of hydrophilic drug 5-fluorouracil (5-FU) across skin. FTIR spectroscopy showed that the T2 peptide interacted with skin lipids to enhance the skin penetration. Pretreating the skin with T2 peptide enhanced the partitioning of small molecules with different lipophilicities (5-FU, fluorescein isothiocyanate, and rhodamine 123 hydrochloride) into skin. Fluorescence studies showed that T2 peptide enhanced the diffusion of these molecules into intercellular lipids of SC and thus enhanced the penetration into the skin. Histidine at the c-terminus of T2 peptide was identified to be critical for the skin penetration enhancement. T2 peptide interacted with skin lipids to cause skin penetration enhancement. The study identified a novel, safe, and noninvasive peptide to improve the skin penetration of drugs without chemical conjugation.

Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors

Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors.

A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.

Molecular Changes in the Vasculature of Injured Tissues

We have explored molecular specialization of the vasculature of regenerating wound tissue in the skin and tendons to identify a different repertoire of markers from that obtained by studying tumor vasculature. We screened a phage-displayed peptide library for peptides that home to wounds in mice and identified two peptides that selectively target phage to skin and tendon wounds: CARSKNKDC (CAR) and CRKDKC (CRK). CAR is homologous to heparin-binding sites in various proteins and binds to cell surface heparan sulfate and heparin. CRK is similar to a segment in thrombospondin type 1 repeat. Intravenously injected CAR and CRK phage, as well as fluorescein-labeled CAR and CRK peptides, selectively accumulated at wound sites, where they partially co-localized with blood vessels. The CAR peptide showed a preference for early stages of wound healing, whereas the CRK favored wounds at later stages of healing. The CAR peptide was internalized into the target cells and delivered the fluorescent label into the cell nuclei. These results identify new molecular markers in wound tissues and show that the expression of these markers in wound vasculature changes as healing progresses. The peptides recognizing these markers may be useful in delivering treatments into regenerating tissues.

Mast Cell-restricted Tryptases: Structure and Function in Inflammation and Pathogen Defense

Mast cells (MCs) are highly specialized immune cells present in mammals and in lower organisms that predate the development of adaptive immunity. The strong evolutionary pressure to retain MCs for >500 million years suggests critical roles for these cells in our survival. In support of this conclusion, no human has been identified to date that lacks MCs, despite the adverse roles of MCs in systemic anaphylaxis and varied inflammatory disorders. MCs express numerous lineage-restricted neutral proteases, and four members of the chromosome 17A3.3 family of tryptases are preferentially expressed in mouse MCs. The anatomical location of MCs at host-environment interfaces has raised the possibility that some of these enzymes are evolutionally conserved because they are needed for combating infectious organisms. Here we review recent insights into the structure and function of MC tryptases in inflammation and host defense against bacteria and other infectious organisms.

Angiogenesis induced by novel peptides selected from a phage display library by screening human vascular endothelial cells under different physiological conditions

Angiogenesis is a process modulated by several endogenous vascular growth factors as well as by oxygen conditions. For example VEGF failed to induce useful therapeutic angiogenesis in clinical trials. We used a combinatory phage display peptide library screening on human umbilical endothelial cells under normoxia and hypoxia conditions in order to identify novel peptides that bind endothelial cells. The identified peptides induced angiogenesis as demonstrated by endothelial cell proliferation, migration and tube formation. Injection of peptides into the ears of mice resulted in increased numbers of blood vessels. Peptides did not induce VEGF receptor gene expression indicating a possible VEGF unrelated mechanism.

Lymphatic Zip Codes in Premalignant Lesions and Tumors

Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention.

Characterisation of an autoreactive conformational epitope on GAD65 recognised by the human monoclonal antibody b78 using a combination of phage display, in vitro mutagenesis and molecular modelling

Autoantibodies to the diabetes autoantigen, the 65 kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model. Alanine mutants of GAD65 based on deduced contact residues were examined for binding with b78 and control sera. Mutation of 524SRL 526, 572DF 573 and 498KPQ 500 reduced reactivity of b78 with mutant GAD65 > 50%. Molecular dynamics indicated that mutation of 498KPQ 500 caused structural changes that could account for effects of this mutation. Thus phage display in combination with molecular modelling identified contact residues within a highly conformational epitope for mAb b78 in the C-terminus of GAD65. These techniques should have broad applicability to definition of epitope structure.

Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides: THE EXAMPLE OF SHIGELLA FLEXNERI 5A

Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites. This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides by peptides and short oligosaccharides.

Vascular Homing Peptides with Cell-Penetrating Properties

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular heterogeneity in the blood vessels of individual normal tissues and shown that pathological lesions put their signature on the vasculature. In tumors, both blood and lymphatic vessels differ from normal vessels. Moreover, the molecular changes in the vasculature parallel progression in tumor development, hence making the vessels in premalignant lesions distinguishable from normal vessels and from the vessels in malignant tumors of the same tissue. Some of the tumor-homing peptides penetrate into tumor endothelial cells (and tumor cells), but not into endothelial cells in normal tissues or other normal cells. Thus, these cell-penetrating peptides are cell type-specific. Peptides that home to tumor vasculature have been shown to be useful in directing therapeutic agents to experimental tumors. The cell penetrating peptides may be particularly useful in drug delivery because they can take their payload inside the target cells and even into a specific subcellular organelle such as the nucleus.

Application of laser capture microdissection to phage display peptide library screening

When identifying important regulatory genes using methods such as phage display peptide library screening it is critical to select such peptides from cells and tissues in their native state. Here, we report a novel approach to screen tumors using phage display and laser capture microdissection (LCM). A phage peptide library was screened directly on fresh oral tumor tissue, such that specifically bound peptides were selected from fresh tumor cells in the native tissue state. Tissue processing conditions were modified to ensure the survival of the bacteriophage. Our results demonstrate that live phage-peptide conjugates can be recovered from laser capture microdissected cells in a form suitable for additional cycles of amplification. Thus, LCM will be a valuable adjunct to phage display studies.

Vascular zip codes in angiogenesis and metastasis.

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular differences in the blood vessels of individual normal tissues. Pathological lesions also put their signature on the vasculature; in tumours, both blood and lymphatic vessels differ from normal vessels. The changes that characterize tumour blood vessels include selective expression of certain integrins. Peptides isolated by in vivo phage display for homing to tumours have been shown to be useful in directing therapeutic agents to experimental tumours. The targeting can enhance the efficacy of the therapy while reducing side effects. Phage screening has also revealed lung-specific vascular markers that promote tumour metastasis to the lungs by mediating specific adherence of tumour cells to the lung vasculature. These phage-screening studies have revealed a previously unsuspected degree of vascular specialization and provide potentially useful guidance devices for targeted therapies.

Selection of peptide ligands binding to the basolateral cell surface of proximal convoluted tubules

Recently, we have reported a novel approach of screening phage-display peptide libraries on microdissected intact renal tubular segments and identified an RGD-containing peptide ligand that specifically binds to the basolateral membrane of cortical collecting ducts (CCD). However, screening phage libraries on proximal convoluted tubules (PCT) did not yield a tubule segment-specific ligand. Here, we describe the successful modification of our previously developed phage-display approach and the identification of two distinct ligands that bind specifically to receptors expressed at the basolateral membrane of PCT. Ex vivo screening of phage-display peptide libraries for specific ligands was adapted for PCT. The previously developed method was significantly extended by applying it to a distinct tubular segment, varying the number of rounds of biopanning and incubating phage libraries with absorber cells prior to biopanning. Binding specificity and cellular localization of selected peptide-displaying phage or the corresponding synthetic peptide were analyzed using various epithelial cell lines as well as competition assays and confocal immunofluorescence microscopy. Screening phage-display peptide libraries, depleted of ligands binding to ubiquitously expressed receptors by preincubation with HEK-293 cells, led to the identification of two PCT-specific ligands. Phage expressing peptides with the consensus sequence GV(K/R)GX3(T/S) or RDXR mediated 15-fold and 13-fold higher binding to PCT than control phage, and binding to PCT was 13-fold and 21-fold higher than binding to CCD, respectively. Neither phage mediated significant binding to various epithelial cell lines, and binding of both ligands was abolished by the addition of the corresponding synthetic peptide. Immunofluorescence experiments revealed a submembrane localization of both ligands upon incubation with PCT. Exploiting the versatility of phage-display and biopanning allowed the identification of two distinct peptide ligands that bind specifically to the basolateral membrane of PCT. Tubule segment-specific ligands, such as the described PCT ligands, may be useful for the analysis of cell-extracellular matrix interactions and may contribute to the development of new therapeutic strategies for renal diseases.

Identification of receptor ligands by screening phage-display peptide libraries ex vivo on microdissected kidney tubules.

A novel method to identify receptor ligands for defined renal tubular segments has been developed. Ex vivo screening of phage-display peptide libraries on isolated intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD) allowed the direct access of phage to the basolateral surface of tubular epithelial cells. Two distinct peptide motifs were selected for CCD and PCT, indicating differential expression of some membrane receptors on the basolateral surface of defined kidney tubule segments. Using the linear peptide motif ELRGD(R/M)AX(W/L), recovered from freshly isolated rat CCD, mediated 16-fold selectivity of phage binding to CCD compared with PCT. Binding to CCD was 39-fold higher than that of a random control phage. Binding and subsequent internalization of phage, most likely by an integrin-mediated endocytosis pathway, was abolished by the addition of the corresponding synthetic peptide. Furthermore, the results demonstrate that presentation and flanking amino acids determine the specific binding properties of RGD ligands to their putative integrin receptors. The results emphasize the need of a native cell system for the identification of renal epithelial cell surface ligands. Such ligands are of potential relevance for the analysis of interactions between extracellular matrix and kidney tubules or for the development of improved vectors for kidney-specific drug delivery or gene transfer.