High-throughput identification of phage-derived imaging agents

The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of "hits" obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.     

噬菌体抗体库筛选技术

在过去的20年中,噬菌体抗体库筛选技术被广泛的应用于抗体筛选、疾病治疗,临床诊断以及基础研究之中。在该技术的应用过程中,快速有效的筛选出最适合的单克隆抗体并进行可靠和高效的数据管理和分析是十分重要的。文中总结了近年来噬菌体抗体库的高通量筛选方法并且对数据管理做了简要介绍。     

Phage display for engineering and analyzing protein interaction interfaces

Phage display is the longest-standing platform among molecular display technologies. Recent developments have extended its utility to proteins that were previously recalcitrant to phage display. The technique has played a dominant role in forming the field of synthetic binding protein engineering, where novel interfaces have been generated from libraries built using antibody fragment frameworks and also alternative scaffolds. Combinatorial methods have also been developed for the rapid analysis of binding energetics across protein interfaces. The ability to rapidly select and analyze binding interfaces, and compatibility with high-throughput methods under diverse conditions, makes it likely that the combination of phage display and synthetic combinatorial libraries will prove to be the method of choice for synthetic binding protein engineering for broad applications.     

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.     

Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.

A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.     

Analysis of interactions of signaling proteins with phage-displayed ligands by fluorescence correlation spectroscopy

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.     

Phage-display as a tool for quantifying protein stability determinants.

To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display libraries to experimentally address questions that have generally been addressed in silico, either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on beta-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have analyzed the energetic contributions of cross-strand side chain-side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addressing biophysical questions.     

Phage Peptide Libraries

Filamentous phage particles have been central in the construction of libraries displaying vast numbers of random peptides. These random peptides can be antigenically presented as fusions to coat proteins III and VIII of the phage. The isolation of ligate-reactive phage from an immense background of nonspecific phage is achieved by the biopanning process. Enrichment of reactive phage relative to unreactive phage occurs with alternate rounds of affinity selection to the desired molecular target and amplification of the specifically bound phage. This allows the isolation of rare binding species contained in the phage peptide libraries. Each phage particle contains the information in its genome pertaining to the type of random peptide insert displayed. Hence, the identification of binding motifs displayed on ligate-reactive phage is revealed by sequencing the relevant insert site in the phage genome. Phage peptide libraries have been used to isolate ligands to an array of protein ligates. The libraries have proved particularly effective in defining the binding sites of monoclonal antibodies and to some extent polyclonal sera. The analysis of the peptide insert sequences of a number of different clones of antibody binding phage can reveal a great deal about the nature and restriction of the amino acid residues critical for the antibody–antigen interaction.     

Generation of bioactive peptides by biological libraries

Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.     

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.     

High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization,Binding and Tumor Targeting

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with ⁶⁴Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.     

Comparative Analysis of the Efficiency of the Binding of Phage Particles Displaying Tumor-Targeting Peptides to Cancer and Healthy Brain Cells

Applied Biochemistry and Microbiology - The efficiency of the binding of tumor-targeting phage particles displaying tumor-targeting peptides, which were obtained with phage-display technology, to...     

Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

Phages from landscape libraries as substitute antibodies

In 'landscape' phage, as in traditional phage-display constructs, foreign peptides or proteins are fused to coat proteins on the surface of a filamentous phage particle. Unlike conventional constructs, however, each virion displays thousands of copies of the peptide in a repeating pattern, subtending a major fraction of the viral surface. The phage body serves as an interacting scaffold to constrain the peptide into a particular conformation, creating a defined organic surface structure ('landscape') that varies from one phage clone to the next. By testing landscape libraries with three representative antigens (streptavidin from the bacterium Streptomyces avidinii, avidin from chicken egg white and beta-galactosidase from Escherichia coli) we have shown that landscape phages may be used as a new type of substitute antibodies-filaments that can bind protein and glycoprotein antigens with nanomolar affinities and high specificity. In many ways these substitute antibodies are more convenient than their natural immunoglobulin counterparts.     

Screening phage-displayed combinatorial peptide libraries

Among the many techniques available to investigators interested in mapping protein-protein interactions is phage display. With a modest amount of effort, time, and cost, one can select peptide ligands to a wide array of targets from phage-display combinatorial peptide libraries. In this article, protocols and examples are provided to guide scientists who wish to identify peptide ligands to their favorite proteins.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

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.     

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.     

Selection of stably folded proteins by phage-display with proteolysis.

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.     

Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library.

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.