Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.     

Immunogenically fit subunit vaccine components via epitope discovery from natural peptide libraries

Antigenic peptides that bind pathogen-specific Abs are a potential source of subunit vaccine components. To be effective the peptides must be immunogenically fit: when used as immunogens they must elicit Abs that cross-react with native intact pathogen. In this study, antigenic peptides obtained from phage display libraries through epitope discovery were systematically examined for immunogenic fitness. Peptides selected from random peptide libraries, in which the phage-displayed peptides are encoded by synthetic degenerate oligonucleotides, had marginal immunogenic fitness. In contrast, 50% of the peptides selected from a natural peptide library, in which phage display segments of actual pathogen polypeptides, proved very successful. Epitope discovery from natural peptide libraries is a promising route to subunit vaccines.     

DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.     

Phage display of random peptide libraries: applications, limits, and potential

The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.     

Phage protein-targeted cancer nanomedicines

Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review.     

Selection of proteins and peptides from libraries displayed on filamentous bacteriophage

This article attempts to review recent developments in the rapidly developing field of phage display libraries. The current state of peptide, antibody, and cDNA libraries, as well as current and future applications of phage display libraries are discussed. The main focus of the article is on the methods for selecting binding ligands against targets in a variety of different formats. These include solid phase and in-solution selection methods, and the strategies used to select for higher affinity, and binding ligands ampure and cellular target proteins.     

mRNA display: ligand discovery,interaction analysis and beyond

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.     

In vivo phage display and vascular heterogeneity: implications for targeted medicine

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics.     

An ephrin mimetic peptide that selectively targets the EphA2 receptor

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K(D) values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.     

Biodistribution of filamentous phage peptide libraries in mice

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens. Phage selections in animals have been performed for very short times without optimization for biodistribution or clearance rates to a particular organ. It is hypothesized that peptides that home to a desired antigen/organ can be obtained from in vivo phage experiments by optimization of incubation times, phage extraction and propagation procedures. To accomplish this goal, one must first gain a better understanding of the in vivo biodistribution and rate of clearance of engineered phage peptide display libraries. While the fate of wild type phage in rodents has been reported, the in vivo biodistribution of the commonly used engineered fd-tet M13 phage peptide display libraries (such as in the fUSE5 vector system) have not been well established. Here we report the biodistribution and clearance properties of fd-tet fifteen amino acid random peptide display libraries in fUSE5 phage in three common mouse models employed for drug discovery - CF-1, nude, and SCID mice.     

Phage presentation

There has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins. Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non-proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies. The display of proteins or their functional domains provides a system for the analysis of structure-function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties. The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by-pass hybridoma technology in the generation of monoclonal antibodies.     

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on m13 phage

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.     

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.     

Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides

Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.     

Novel peptide inhibitors of human kallikrein 2

Human kallikrein 2 (hK2) is a serine protease produced by the secretory epithelial cells in the prostate. Because hK2 activates several factors participating in proteolytic cascades that may mediate metastasis of prostate cancer, modulation of the activity of hK2 is a potential way of preventing tumor growth and metastasis. Furthermore, specific ligands for hK2 are potentially useful for targeting and imaging of prostate cancer and for assay development. We have used enzymatically active recombinant hK2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. Using libraries expressing 10 or 11 amino acids long linear peptides, we identified six different peptides binding to hK2. Three of these were shown to be specific and efficient inhibitors of the enzymatic activity of hK2 toward a peptide substrate. Furthermore, the peptides inhibited the activation of the proform of prostate-specific antigen by hK2. Amino acid substitution analyses revealed that motifs of six amino acids were required for the inhibitory activity. These peptides are potentially useful for treatment and targeting of prostate cancer.