Combinatorial Phage Display Library Research Articles

Selection and Characterization of Phage Miniantibodies to Actins of Different Origin

Single-chain miniantibodies (scFv's) to actin were obtained by the phage display method. A naive combinatorial phage display library of murine scFv's (containing 2x10(8) independent recombinant clones) was used to select miniantibodies. After three rounds of selection two clones producing miniantibodies to chicken smooth muscle actin with affinity constants of 1.4x10(7) and 1.2x10(6) M(-1) were chosen. The isolated miniantibodies could specifically detect various plant and animal actins.

Humanization of chicken monoclonal antibody using phage-display system

We describe a simple method for humanizing chicken monoclonal antibody (mAb). Humanization of mAbs by simple CDR-grafting often results in loss of affinity because certain framework residues of the antibody variable regions can participate in antigen–antibody interaction. In this study, humanization of chicken mAbs was achieved by CDR-grafting, followed by framework fine-tuning using a chicken phage-displayed mAb, phAb4-31, as a model antibody. In order to fine-tune the framework, we used the phage-displayed combinatorial library with permutation of important framework residues. After panning the humanized library, the “most humanized” variants were selected and analyzed for antigen-binding activity. All of these clones retained affinity comparable to the parental chicken mAb. These results suggest that chicken mAbs can easily be humanized, and thus humanized chicken mAbs may be practically applied as therapeutic agents.

966. AAV Delivered Single-Chain Antibodies as Passive Immuno-Therapy for Prion Disease

Top of pageAbstract Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a normal cellular protein (PrPc) to an abnormal disease causing conformation designated PrPsc. Efforts to use antibodies to alter the rate of conversion of PrPc to PrPsc suggest it to be a plausible therapeutic strategy. Notably, during the normal progression of disease the host organism mounts no detectable immune response to alter the process of PrPsc accumulation. This is hypothesized to be due to the low antigenicity of PrPc and host tolerance to a self-polypeptide. We intend to circumvent the lack of a host-based immune response to impede the conversion of PrPc using novel and previously characterized PrPc-specific single-chain variable fragment (scFv) antibodies that will be utilized in an AAV-based passive immuno-therapy. Several novel PrPc-specific antibodies have been obtained from a na|[iuml]|ve human combinatorial phage display library. Antibody D18 has been previously characterized with the ability to clear PrPsc from chronically infected cell-lines. Gross motor coordination of immunized challenged mice is being monitored by Rotarod performance. Data from ongoing evaluation of these vaccinated animals will be presented.

In vivo biotinylated proteins as targets for phage-display selection experiments

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus (pMCSG16 and 15) via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase. To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, also contain a ligase-independent cloning site (LIC), which permits up to 100% cloning efficiency. The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries. From selections using the N-terminally biotinylated version of one target protein, a peptide ligand ( K d =9 μ M) was recovered that bound in a format-dependent manner. To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries.

222. HSV Amplicon Expression of Single Chain Antibodies Directed Against α-Synuclein Conformers

Parkinson's disease (PD) is a progressive neurodegenerative disorder which targets the nigrostriatal system and is triggered by disparate mechanisms. The hallmark pathobiologic feature of PD is the formation of intracytoplasmic inclusions known as Lewy bodies. Lewy bodies are composed of aggregated proteins, most notably α-synuclein. The selective insult to the dopamine (DA) neuron is posited to involve oxidative injury mediated in part through participation of DA adducts made onto α-synuclein (α-syn). Oxidative stress and DA-derived orthoquinone adducts form onto α-syn promoting the formation of misfolded protein. We hypothesize that therapies specifically designed to interact and reduce toxic α-syn conformers will attenuate disease progression. We are pursuing a gene therapy approach whereby conformation-specific humanized single-chain Fvs (scFvs) are expressed and intended to interfere with misfolded α-syn. Utilizing various conformers of purified α-syn to screen a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions we have identified conformation-specific α-syn-specific scFvs. These scFvs have been efficiently expressed in mammalian cells following transduction with an HSV amplicon vector (HSVscFvs). The effect of transduction and expression of intracellular scFvs, termed intrabodies, within stable cell lines overexpressing α-syn is under investigation. HSVscFvs directed against α-syn afford us the opportunity to further investigate the role of α-syn aggregation in the pathogenesis of PD.

554. A Candidate SARS-Associated Coronavirus Vaccine Elicits Broad Immunity in Monkeys

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a normal cellular protein (PrPc) to an abnormal disease causing conformation, designated PrPsc. Efforts to use antibodies to alter the rate of conversion of PrPc to PrPsc suggest it to be a plausible therapeutic strategy. Notably, during the normal progression of disease the host organism mounts no detectable immune response to alter the process of PrPsc accumulation. This is hypothesized to be due to the low antigenicity of PrPc and host tolerance to a self-polypeptide. We intend to circumvent the lack of a host-based immune response to impede the conversion of PrPc using novel PrPc-specific single-chain variable fragment (scFv) antibodies that will be utilized in a viral-based passive immuno-therapy. Several PrPc-specific antibodies have been obtained from a naive human combinatorial phage display library. Epitope mapping has been performed on our scFvs using an ELISA based peptide array for linear epitope determination. We have also investigated the clearance of scFvs from a thalamic injection and determined the elimination half-life (T1/2).

Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library

Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications. In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library. We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site. Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original. In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

Humanization of antagonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library.

Anti-4-1BB (CD137) monoclonal antibody (mAb) has been reported to suppress immune responses and to have the potential for use as a therapeutic agent to block autoimmune diseases. Previously, the authors prepared an antagonistic anti-human 4-1BB (CD137) mAb, BBK2. Here the authors report the humanization of BBK2 using a phage display library. Four humanized single-chain Fv (scFv) fragments were selected from a combinatorial library expressing a phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb and to have affinities higher than those of the original. Both the soluble and bound forms of the humanized scFv suppressed the proliferation of human peripheral blood mononuclear cells, similar to the original mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

The Mapping and Reconstitution of a Conformational Discontinuous B-cell Epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.

Cassette vectors for conversion of Fab fragments into full-length human IgG1 monoclonal antibodies by expression in stably transformed insect cells.

Phage display technology allows for the production and rapid selection of antigen-specific, Fab antibody fragments. For purposes of immune therapy, though, complete antibodies that retain the Fc domain are often required. In this regard, we designed cassette vectors for converting human Fab fragments selected from combinatorial phage display libraries into full-length IgG(1) monoclonal antibodies (MAbs). Two expression vectors, pIEI-Light and pIEI-Heavy, were engineered to contain respective light- and heavy-chain human signal sequences downstream of the baculovirus immediate early gene promoter, IEI. Vector pIEI-Heavy also contains the coding region for each of the human IgG(1) constant domains. To generate complete antibody genes, the cassette vectors possess convenient restriction enzyme sites for rapid in-frame cloning of coding regions for full-length light chains in pIEI-Light and for the heavy-chain variable domains in pIEI-Heavy of Fab fragments. Using these constructs and a method that allows for stable transformation of insect cells, complete light- and heavy-chain genes can be inserted into the insect cell genome and subsequently expressed under the control of the baculovirus IEI promoter. This cassette vector system was used to generate stably transformed insect cells that continuously secreted functional full-length, IgG(1) MAbs. The expressed antibodies exhibited light and heavy chains of the appropriate molecular sizes and retained the ability to bind antigen. We conclude that our cassette vectors could serve as valuable tools for generating human IgG(1) antibodies.

Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.

Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain.

Hantaan virus (HTNV) in the Hantavirus genus, family Bunyaviridae, is the major cause of severe hemorrhagic fever with renal syndrome (HFRS). We prepared a combinatorial phage display library of human Fabs to HTNV from RNA extracted from the blood lymphocytes of a convalescent HFRS patient. We selected two G1 glycoprotein-specific clones and one nucleocapsid protein (N)-specific clone from the Fab library for further studies. The human Fab antibodies were converted to IgG form in baculovirus/insect cells system by using cassette vectors that we developed earlier. Characterization of the recombinant antibodies revealed that the two G1-specific IgGs, could bind to and neutralize HTNV but not Seoul virus (SEOV). The N-specific IgG did not neutralize either HTNV or SEOV. Sequence analysis revealed that the two G1-specific clones differed by only one predicted amino acid in their complementarity determining regions, CDR3. Epitope mapping studies were carried out with one of the two G1-specific clones and synthetic peptides representing portions of HTNV G1. Results indicated that the recombinant antibody recognizes the core amino acid sequence LTKTLVIGQ, which is found near the C-terminus of HTNV G1. These results are the first to define a neutralizing epitope on the G1 protein of HTNV using an antibody derived from an HFRS patient.

Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library

Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.

Quantitative Assessment of Peptide Sequence Diversity in M13 Combinatorial Peptide Phage Display Libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based Mutagenesis

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.

Development of Novel Tumor Imaging Agents with Phage-Display Combinatorial Peptide Libraries

Rationale and Objectives Current radiologic methods do not provide sufficient information for unambiguous diagnosis and prognosis of cancer. The present investigation sought to address this deficiency by developing a system for designing novel small molecules targeted against tumor-specific molecules for use as radionuclide imaging agents. Materials and Methods Part of a tumor-specific receptor, purified recombinant epidermal growth factor receptor (EGFR), variant III, extracellular domain (rEGFRvIII-ecd), was used as the target in the selection of EGFRvIII-specific peptide ligands from random peptide bacteriophage (phage) display libraries. After three rounds of screening, phage isolates were tested for binding affinity with an enzyme-linked immunosorbent assay. Positive phage were sequenced, and the peptides were synthesized and tested for binding affinity with a surface plasmon resonance assay. Results Affinity screening identified 49 peptide-expressing phage that showed enhanced binding to the variant receptor compared with wild-type EGFR. Free peptides from the two phage isolates exhibiting the most favorable binding were tested for target binding. One of these demonstrated a binding affinity for rEGFRvIII-ecd in the 30-nmol/L range. Conclusion These data suggest that phage display libraries may be very useful in the design of novel, high-affinity tumor imaging agents.

Synthetic Peptides Identified from Phage-displayed Combinatorial Libraries as Immunodiagnostic Assay Surrogate Quality-Control Targets

Quantitative immunohistochemical (IHC) assays currently lack optimal reference quality-control material for cellular protein targets. To address this problem, we identified peptides that mimic the site on the native analyte to which the primary (monoclonal) antibody binds and used them as surrogate peptide controls. We identified peptide candidates from a combinatorial peptide phage-display library that mimic the epitope for the 1D5 estrogen receptor (ER) monoclonal antibody (mAb). The peptide inserts of the phage clones were sequenced. Several phage-encoded peptides were then synthesized and analyzed for affinity and specificity. We identified phage clones that specifically bound to the ER 1D5 mAb. The binding was specific, in that the phage clones did not bind to two other isotype-matched mAbs. Their ability to bind the ER 1D5 mAb was related to the presence of a consensus sequence. Binding analysis revealed a K(d) of 8.3 x 10(-8) mol/L. The peptide was not recognized by any of 15 other mAbs commonly used for clinical IHC testing. Moreover, the peptide was able to inhibit the binding of ER 1D5 mAb to native ER, indicating that the peptide bound to ER 1D5 mAb at or close to the antigen-binding site. Surrogate peptide controls behave like the native analyte in terms of affinity and specificity. This technology may be especially useful when the native analyte is in short supply.

Isolation and characterization of recombinant human IgE antibodies from pediatric egg allergic patients the combinatorial phage display library approach

Isolation and characterization of recombinant human IgE antibodies from pediatric egg allergic patients the combinatorial phage display library approach

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Selection of phage-display library peptides recognizing ethanol targets on proteins

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein–ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein–protein interactions. A general screening, M13-based library with 10 9 random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage’s minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein–ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein–ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.