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PubMed1999

Epitope discovery using monoclonal antibodies and phage peptide libraries.

Phage display is a biological system which facilitates the cloning and rapid selection of peptides from large combinatorial libraries. In comparison to the chemical combinatorial approach, the advantages of phage display lie in its simplicity and replicability. While phage display has many diverse applications, this review will focus on the use of phage peptide libraries to discover epitopes recognised by monoclonal antibodies. As monoclonal antibodies are useful tools for the detection of proteins and for the investigation of molecular interactions, the identification of their epitopes will serve to elucidate the structure and function of proteins, as well as aid in the discovery of new drugs and the development of vaccines.

Amino Acid Sequence↗
PubMed2001

Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.

Mimotopes of a tumor-associated T cell epitope were determined using randomized and combinatorial peptide libraries and a CD8(+) T cell clone specific for the cutaneous T cell lymphoma cell line MyLa. Antigen recognition by this clone was found to be HLA-B8 restricted. More than 80 % of HLA-matched patients with cutaneous T cell lymphoma had mimotope-specific CD8(+) T cells in their peripheral blood. Mimotope-specific T cells isolated and expanded from a patient lysed MyLa cells in in vitro assays thus demonstrating their cytolytic capacity and tumor specificity. Mimotope vaccination of a patient without detectable mimotope-specific T cells induced frequencies of these cells of up to 1.82 % of the peripheral blood CD8(+) T cells.

Amino Acid Sequence↗
PubMed2005

[Study on screening mimicking epitope of ochratoxin A from phage display peptide library].

OBJECTIVE: To screen phage particles capable of mimicking ochratoxin A and establish immunoassay for ochratoxin A with it. METHODS: An anti-ochratoxin A monoclonal antibody was used as ligand. Biopanning was done to screen the mimicking epitope from a phage random 7 peptide library. This library was displayed as a fusion protein with the coat protein III of filamentous phage M13. The positive clones were identified by ELISA, the inserted amino sequences were deduced by DNA sequencing. RESULTS: After four rounds of panning, 11 positive clones can bind to the antibody, and the binding can be blocked the free ochratoxin A. The common amino sequence of the mimicking epitope is IRPMVXX. A competitive ELISA immunoassay was established with clone P2, the linear range of the inhibition curves is between 200 pg/ml and 8000 pg/ml, the detecting limitation is 150 pg/ml. CONCLUSION: The phage display technique can be successfully applied to screen the mimicking epitope of ochratoxin A. The acquired phages may be used as the surrogate of the toxin to establish the immunoassay.

Antibodies, Monoclonal↗
PubMed1993

Multiple sequence analysis: pool sequencing of synthetic and natural peptide libraries.

Sequence analysis by automated Edman degradation has been one of the most powerful tools for the characterization of peptides for many years. Its sensitivity allows experiments in the low picomole range, and its results provide complete information about the primary structure of proteins and peptides. A new method in protein and peptide sequencing has been developed which allows for the direct application of automated Edman degradation to the analysis of peptide mixtures. With the aid of multiple sequence analysis, first, sequence motifs of isolated, naturally processed peptides binding to MHC molecules have been defined. Second, the method proved to be a valuable and reliable tool for the characterization of synthetic peptide libraries, and third, the specificity of proteases has been determined in a fast and efficient way.

Amino Acid Sequence↗
PubMed2005

Identification of a deoxyribonuclease I inhibitor from a phage-peptide library.

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity 10(8) and 10(9) for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction (IC50 = 0.1-0.7 microM) and DNA degradation by DNase I (IC50 = 0.8-8 microM). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

Actins↗
PubMed2002

Selection of mimotopes of the cell surface adhesion molecule Mel-CAM from a random pVIII-28aa phage peptide library.

The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma. Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells. The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays. For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7. After three panning rounds a population of phages binding to MAd18-5D7 was enriched. Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site. DNA sequences coding for two specific peptide ligands were determined. One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM. Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate. Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments. No such inhibition was observed using a panel of other anti-Mel-CAM antibodies. Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell-cell interaction.

Amino Acid Sequence↗
PubMed2006

Peptide mimotopes selected from a random peptide library for diagnosis of Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a ubiquitous, worldwide infectious agent that causes infectious mononucleosis, affecting >90% of the world's population. Currently, enzyme-linked immunosorbent assay, mostly with purified preparations of EBV cell extracts to capture immunoglobulin M (IgM) antibodies in patients' serum, is used for primary diagnosis. Our objective was to determine whether a small set of peptides could contain sufficient immunogenic information to replace solid-phase antigens in EBV diagnostics. Using monoclonal antibodies, we selected four peptides that mimic different epitopes of EBV from a phage-displayed random peptide library. To assess their diagnostic value, we screened a panel of 62 individual EBV IgM sera for their reactivities with the peptides alone. For all peptides, there was a clear distinction between the EBV-positive and the EBV-negative samples, resulting in 100% specificity. The sensitivities were 88%, 85%, 71%, and 54% for peptides F1, A3, gp125, and A2, respectively. Any combination of peptides increased the sensitivity, indicating that individual peptides react with different subsets of antibodies. Furthermore, when the F1 and the gp125 peptides were coupled to bovine serum albumin and screened against 216 serum samples, there were dramatic improvements in sensitivities (95% and 92%, respectively) and little cross-reactivity with the other peptides encountered during acute viral infections, including rheumatoid factor. This study shows the potential for the use of peptide mimotopes as alternatives to the complex antigens used in current serodiagnostics for EBV infection.

Amino Acid Sequence↗
PubMed1995

Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions.

We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. In developing the system the entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface. A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E. coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin. To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies. We have unambiguously mapped three different antibody epitopes using this method. Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions. This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

Amino Acid Sequence↗
PubMed1997

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries.

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the beta cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Amino Acid Sequence↗
PubMed2006

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library.

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.

Amino Acid Motifs↗
PubMed2006

Molecular diversification in spider venoms: a web of combinatorial peptide libraries.

Spider venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years. The application of technologies derived from genomics and proteomics have led to the discovery of the enormous molecular diversity of those venoms, which consist mainly of peptides and proteins. The molecular diversity of spider peptides has been revealed by mass spectrometry and appears to be based on a limited set of structural scaffolds. Genetic analysis has led to a further understanding of the molecular evolution mechanisms presiding over the generation of these combinatorial peptide libraries. Gene duplication and focal hypermutation, which has been described in cone snails, appear to be common mechanisms to venomous mollusks and spiders. Post-translational modifications, fine structural variations and new molecular scaffolds are other potential mechanisms of toxin diversification, leading to the pharmacologically complex cocktails used for predation and defense.

Amino Acid Sequence↗
PubMed2002

Toward synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ Tumor-reactive T-Cell Ligands: a comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ cytolytic T-lymphocyte clone.

The use of synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) has emerged recently as an alternative approach for the identification of peptides recognized by T lymphocytes. The choice of both the PS-SCL used for screening experiments and the method used for data analysis are crucial for implementing this approach. With this aim, we tested the recognition of different PS-SCL by a tyrosinase 368-376-specific CTL clone and analyzed the data obtained with a recently developed biometric data analysis based on a model of independent and additive contribution of individual amino acids to peptide antigen recognition. Mixtures defined with amino acids present at the corresponding positions in the native sequence were among the most active for all of the libraries. Somewhat surprisingly, a higher number of native amino acids were identifiable by using amidated COOH-terminal rather than free COOH-terminal PS-SCL. Also, our data clearly indicate that when using PS-SCL longer than optimal, frame shifts occur frequently and should be taken into account. Biometric analysis of the data obtained with the amidated COOH-terminal nonapeptide library allowed the identification of the native ligand as the sequence with the highest score in a public human protein database. However, the adequacy of the PS-SCL data for the identification for the peptide ligand varied depending on the PS-SCL used. Altogether these results provide insight into the potential of PS-SCL for the identification of CTL-defined tumor-derived antigenic sequences and may significantly implement our ability to interpret the results of these analyses.

Biometry↗
PubMed2000

Analysis of T cell responses to the beta 2-glycoprotein I-derived peptide library in patients with anti-beta 2-glycoprotein I antibody-associated autoimmunity.

Antiphospholipid syndrome (APS) is an autoimmune disease that accompanies anti-phospholipid antibodies measured as either anti-cardiolipin antibodies (aCL) or lupus anticoagulant. beta(2)-glycoprotein I (beta(2)GPI) is the most common and apparently the best-characterized antigenic target for aCL. To investigate T-cell responses to beta(2)GPI, we stimulated PBMC of 18 APS or systemic lupus erythematosus (SLE) patients carrying anti-beta(2)GPI and 10 healthy controls, using a peptide library covering the beta(2)GPI sequence. We established seven CD4(+) T cell lines reactive with beta(2)GPI peptide. Three of four epitopes for patient-derived T cell lines were p244-264, whereas one T cell line from a control subject also recognized p244-264. Furthermore, there was no tendency for particular HLA class II molecules to present beta(2)GPI peptides. However, cytokine producing patterns were significantly different between T cell lines from patients and those from healthy individuals (p =.028); patients' T cells tend to exhibit higher IL-4 and lower IFN-gamma responses. These T cell lines did not react to beta(2)GPI purified from human plasma. These results indicate that beta(2)GPI-reactive CD4(+) T cells of APS/SLE patients mainly recognize cryptic p244-264 in the context of various HLA class II molecules, and exhibit Th0-Th2-type responses. Our findings may provide a clue to the pathogenesis of APS.

Adult↗
PubMed1998

Evaluation of resins for on-bead screening: a study of papain and chymotrypsin specificity using PEGA-bound combinatorial peptide libraries.

TentaGel, ArgoGel and PEGA resins were evaluated for on-bead biological screening, using a fluorescently-labelled peptide attached to each and assayed for papain activity. Peptide attached to PEGA was cleaved in near quantitative yield at the expected sites, whilst an identical sequence on TentaGel and ArgoGel beads was hydrolysed in very low yields and nonspecifically on ArgoGel. The compatibility of PEGA with enzymes was further demonstrated by the determination of subsite specificities of papain and chymotrypsin using PEGA-bound peptide libraries, which proved to be similar to those observed in free solution.

Chymotrypsin↗
PubMed2002

A subset-orientated algorithm for minimizing the number of steps required for synthesis of cyclic-peptide libraries.

Libraries of cyclic peptides are being synthesized using combinatorial chemistry for high throughput screening in the drug discovery process. This paper describes the min_syn_steps.cpp program (available at http://www.imb.uq.edu.au/groups/smythe/tran), which after inputting a list of cyclic peptides to be synthesized, removes cyclic redundant sequences and calculates synthetic strategies which minimize the synthetic steps as well as the reagent requirements. The synthetic steps and reagent requirements could be minimized by finding common subsets within the sequences for block synthesis. Since a brute-force approach to search for optimum synthetic strategies is impractically large, a subset-orientated approach is utilized here to limit the size of the search.

Journal Article↗
PubMed2003

Mimotopes of tumor-associated T-cell epitopes for cancer vaccines determined with combinatorial peptide libraries.

Cytotoxic T-cells are the most important effector cells in immune responses against tumors. The identification of tumor-associated epitopes for these cells, therefore, has become a key aspect of the development of cancer vaccines. Here, we describe a new approach to the determination of tumor-associated T-cell epitopes which employs combinatorial peptide libraries with singly defined sequence positions in a randomized context. The analysis of the responses of a T-cell clone to these libraries yields the amino acid constituents of the epitope which can be combined to obtain mimotopes that are suitable as vaccine antigens for the induction of tumor-specific responses.

Amino Acid Sequence↗
PubMed1996

Use of combinatorial peptide libraries to construct functional mimics of tumor epitopes recognized by MHC class I-restricted cytolytic T lymphocytes.

Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.

Amino Acid Sequence↗