Phage-displayed Random Peptide Library Research Articles

Highlights of This Issue

Poor localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. To overcome these limits, Shin and colleagues designed a novel solid tumor–penetrating antibody format, mAb-A22p, by genetic fusion of high-affinity neuropilin-targeting A22p peptide to the C-terminus of the heavy chain of the conventional mAbs, such as anti-EGFR cetuximab and anti-Her2 trastuzumab. The mAb-A22p antibodies showed much better tumor homing, extravasation from the vessels, and tumor tissue penetration, resulting in more potent antitumor efficacy, compared with the parent mAbs. This study indicates that mAb-A22p is a superior format for solid tumor–targeting therapeutic antibodies.Mcl-1 represents an important survival factor for pancreatic cancer. In this study, Abulwerdi, Liao, and colleagues reported the preclinical data for a novel, selective small molecule Mcl-1 inhibitor, UMI-77, that targets the BH3-binding groove of Mcl-1. UMI-77 potently inhibits in vitro and in vivo pancreatic cancer growth by antagonizing Mcl-1 function and inducing Bax/Bak-dependent apoptosis. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. These data provide evidence of Mcl-1 as a potential therapeutic target for pancreatic cancer and UMI-77 is currently being evaluated in combination with chemotherapy and radiotherapy.One of the major mechanisms underlying antimicrotubule drug (AMD) resistance involves acquired inactivation of the spindle assembly checkpoint (SAC). In this study, Miao, Wu, and colleagues report that synuclein gamma (SNCG), highly expressed in cancer cells but not in normal epithelium, is sufficient to induce AMD resistance in breast cancer through inhibiting the activity of spindle checkpoint kinase BubR1. SNCG-overexpressed cancer cells have a reduced ability to initiate and maintain proper mitotic arrest, which leads to AMD resistance by premature exit, before cells initiate apoptosis. These findings provide the basis for targeting SNCG in overcoming AMD resistance in cancer treatment.The UN1 monoclonal antibody (mAb) recognized CD43 glycoforms expressed in lymphoblastoid T-cell lines and solid tumors, and not in their normal counterpart. Here, Tuccillo, Palmieri, and colleagues demonstrated that UN1 mAb was endowed with ADCC activity in vitro and antitumor activity in vivo. By screening a phage-displayed random peptide library, they also identified the phagotope 2/165 as a mimotope of the UN1 antigen, which was able to elicit specific antibodies against the UN1/CD43 epitope in mice. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes and indicate the potential of cancer-associated CD43 as a target for immunotherapy.

Cancer-Associated CD43 Glycoforms as Target of Immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.

Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched 469KTPAEAR475 of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.

Mimotope-based prediction of B-cell epitopes.

Mimotopes are peptides mimicking epitopes on the corresponding antigen. They can be obtained via panning the phage-displayed random peptide library against the corresponding monoclonal antibody or specific sera. Besides mimotopes however, the experimental results also include all kinds of unwanted sequences called "target-unrelated peptides," which often interfere with the subsequent experimental and computational analyses. Nevertheless, the prediction of B-cell epitopes based on the experimental result of phage display has shown to be a promising and reliable strategy with acceptable precision. In this chapter, we summarize mimotope-based prediction of B-cell epitopes under three conditions and focus on protocols and tips for retrieving, cleaning, and decoding the data from phage display technology.

IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy

BackgroundDevelopment and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational epitopes. ObjectiveThe aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. MethodsPolyclonal sera from three individual patients, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library. Resulting epitope-mimicking sequences were aligned for identification of consensus sequences and localised on the surface of the Ara h 1 molecule by a computer-based algorithm. ResultsAll epitope-mimicking sequences identified were found to correspond to conformational epitopes. Each individual patient had his/her own distinct IgE as well as IgG4 epitope recognition profile, though some important IgE epitopes were common to all patients. In general the IgG4 epitope pattern was more heterogeneous than the IgE pattern, did not coincide with IgE epitopes and had a lower affinity than IgE. ConclusionsThis study demonstrated the usefulness of the phage-display technology in distinguishing between the epitope pattern of IgE and IgG4, giving detailed information on fine specificity and affinity. Competitive immuno-screening of phage-display random peptide libraries could be a future valuable tool to study the balance and dynamics of the IgE and IgG4 epitope recognition repertoire and provide a diagnostic tool giving information on the associated allergic phenotype.

Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody

The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infection; it is involved in binding to host receptors and contains specific epitopes that elicit neutralizing antibodies. However, most previous studies have not provided detailed molecular information about the functional epitopes on JEV EDIII protein. In this study, we described a monoclonal antibody (mAb 2B4) we produced and characterized by IFA, PRNT, ELISA and Western blot analyses. The results showed that mAb 2B4 was specific to JEV EDIII protein and possessed high neutralization activity against JEV in vitro. Furthermore, we found that the motif, 394HHWH397, was the minimal unit of the linear epitope recognized by mAb 2B4 through screening a phage-displayed random 12-mer peptide library. Using sequence alignment analysis it was found that this motif was highly conserved among JEV strains and was present in West Nile Virus (WNV). Indeed, ELISA data showed that this epitope could be recognized by both JEV-positive swine serum and WNV-positive swine serum. Notably, this linear epitope was highly hydrophilic and was located within the terminal end of a β-pleated sheet of EDIII. An analysis of the spatial conformation supported the possibility of inducing specific antibodies to this epitope. Taken together, we identified 394HHWH397 as an EDIII-specific linear epitope recognized by mAb 2B4, which would be beneficial for studying the pathogenic mechanism of JEV; and mAb 2B4 was also a potential diagnostic and therapeutic reagent.

Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model

Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.

Two Expression Vectors, Designated as pGEX-CDON and pC89S4-CDON, for Producing GST-CDON and pVIII-CDON Fusion Proteins

Deoxynivalenol (DON) mimotope, designated as CDON, is an epitope (CMRPWLQ) immunoscreened from a phage-displayed random peptide library. In order to replace the conjugated toxin with non-toxic recombinant proteins in ELISA, two novel expression vectors, which were designated as plasmid pGEX-CDON and phagemid pC89S4-CDON for producing GST-CDON and pVIII-CDON fusion proteins in E.coli were constructed. After purification, both GST-CDON and pVIII-CDON fusion proteins show good reactogenicity with an anti-DON antibody in a competitive inhibition ELISA test. When GST-CDON was used as coating antigen, the linear range of the competitive inhibition ELISA is from 62ng/ml to 410ng/ml, the linear equation is Y= 186.6-23.87Ln (X), IC50 is 194ng/ml. For pVIII-CDON as coating protein, the linear range of the competitive inhibition ELISA is from 20ng/ml to 470ng/ml, the linear equation is Y = 161.3-25.49Ln (X), R2=0.9962, IC50 is 94ng/ml. ELISA analysis and comparison show the reactogenicity and specificity of pVIII-CDON binding to anti-DON antibody are better than GST-CDON fusion protein. The pVIII-CDON is promising in establishing an ELISA without the use of the toxic mycotoxin conjugate.

Identification of a linear B-cell epitope within the Bluetongue virus serotype 8 NS2 protein using a phage-displayed random peptide library

The NS2 protein of Bluetongue virus (BTV) is an important non-structural protein and plays important roles in viral replication and assembly. In this study, one monoclonal antibody (mAb), 4D4, was raised against BTV8 NS2. Phage display technology was used and identified the consensus binding motif SNYD recognized by mAb 4D4. To define the minimal region required for antibody binding, a panel of synthetic peptides encompassing SNYD derived from the BTV8 NS2 was then used to more specifically define the 4D4 epitope as 149RSNYDV154. Furthermore, amino acid sequence alignments of different BTV serotypes and other orbiviruses suggested that this epitope is highly conserved among the BTV serotypes. The mAb reagent generated in this study may be applied to the development of BTV diagnosis and surveillance programs and the epitope defined here can lead to important insights into how BTV might interact with the sheep's immune system.

Identification of Synthetic Peptides that Inhibit Lipopolysaccharide (LPS) Binding to Myeloid Differentiation Protein-2 (MD-2)

Many studies have suggested that the synergic effect of myeloid differential protein-2 (MD-2) on bacterial lipopolysaccharide (LPS) stimulation of toll-like receptor 4 (TLR4) may be a critical step during the LPS-TLR4 response signaling pathway. We performed a bioinformatic analysis on the MD-2 protein and identified the amino acid sequence NH2-FSKGKYKCV-COOH (K128-132) as a possible key sequence involved in the binding between MD-2 and LPS. We then screened a random phage display peptide library using this sequence as bait in order to identify antagonistic peptides. After 3 rounds of selection, 3 positive clones were identified. All 3 peptides were shown to inhibit, in a dose-dependent manner the production of tumor necrosis factor-α and interleukin-6 in human U937 and THP-1 cell lines as well as human peripheral blood monocytes stimulated by LPS. Only 2 of the 3 peptides were able to bind MD-2 directly as shown by sulfo-SBED biotin label transfer experiments. BALB/C mice were used to estimate the protection of these peptides from LPS challenge, and 2 of the 3 peptides (Lys-Thr-Val-Pro-Asp-Asn-His and Ile-Gly-Lys-Phe-Leu-Tyr-Arg) reduced mortality of the challenged mice from 100% to 53.8%. This study has demonstrated that interfering with the binding between MD-2 and LPS might be a potential therapeutic strategy for treating LPS-induced sepsis, and in doing so has identified 2 potential peptide candidates.

Mapping a Disordered Portion of the Brz2001-Binding Site on a Plant Monooxygenase, DWARF4, Using a Quartz-Crystal Microbalance Biosensor-Based T7 Phage Display

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

Identification of a highly reactive substrate peptide for transglutaminase 6 and its use in detecting transglutaminase activity in the skin epidermis

Mammalian transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent crosslinks between glutamine and lysine residues in proteins. These catalytic reactions play roles in several essential biological processes, including blood coagulation, skin formation, and stabilization of the extracellular matrix. Among the members of this family, factor XIII and TGs 1-5 have been characterized well, but very little is known about the novel members TG6 and TG7. Recently, however, autoantibodies against TG6 were found in a patient with gluten ataxia, a disease caused by enzymatically modified gluten-derived peptides in neuronal cells. To characterize the possible physiological functions of TG6, in this study we screened a phage-displayed random peptide library to find highly reactive glutamine donor substrate peptides. From several candidate peptides, one sequence, designated Y25, appeared to have the highest reactivity. The Y25 sequence also has apparent isozyme specificity when evaluated by incorporation of the labeled glutamine acceptor substrate as a fusion protein with glutathione-S-transferase. Also, the sequence retained high reactivity as well as the isozyme specificity in the peptide form. Analyses with the biotin-labeled and fluorescence-labeled peptides showed TG6 to be an active enzyme and react to specific substrates in the skin, which is consistent with the results of the expression pattern of its transcripts.

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

BackgroundThe gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.Methods and ResultsThis study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched 213SVQYHPL219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that 213SVQYHPL219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.Conclusions and SignificanceWe identified 213SVQYHPL219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

Identification and characterization of antigenic epitope of Staphylococcus aureus ClfA adhesin

Staphylococcus aureus ClfA adhesin is a protective antigen that induces partial immunity against S. aureus infection in mice. To identify the antigenic epitope of ClfA, a monoclonal antibody (mAb) D01 against the recombinant protein was produced by the hybridoma technique. The mAb was used to immunoscreen a random phage-displayed peptide library as the immunogen. After three rounds of biopanning, 41 positive clones were identified. Sixteen phage clones were sequenced and their amino acids were deduced. One mimotope (SKVGIDKRRGTA) showed good match with ClfA adhesin at 383–394 aa and the serum of mice induced by the phage clone clearly recognized ClfA adhesin.

Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

AbstractAbstract 2880 Background:The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods:The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results:Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions:These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures:No relevant conflicts of interest to declare.

Mimotopes selected by biopanning with high-titer HIV-neutralizing antibodies in plasma from Chinese slow progressors

ObjectiveOne approach to identifying HIV-1 vaccine candidates is to dissect the natural antiviral immune response in treatment-naïve individuals infected for over ten years, considered slow progressor patients (SPs). It is suspected that SP plasma has strongly neutralizing antibodies (NAb) targeting specific HIV viral epitopes. MethodsNAbs levels of 11 HIV-1-infected SPs were detected by PBMC-based neutralization assays. To investigate SP NAb epitope, this study used a biopanning approach to obtain mimotopes of HIV-1 that were recognized by SP plasma NAbs. IgG was purified from high-titer NAb SP plasma, and used as the ligand for three rounds of biopanning to select HIV-specific mimotopes from a phage-displayed random peptide library. Double-antibody sandwich ELISA, competitive inhibition assays, and peptide sequence analysis were used to evaluate the characteristics of phage-borne mimotopes. ResultsSPs had significantly more plasma neutralizing activity than typical progressors (TPs) (p=0.04). P2 and P9 plasma, which have highest-titer HIV-NAb, were selected as ligands for biopanning. After three rounds of biopanning, 48 phage clones were obtained, of which 22 clones were consistent with requirement, binding with HIV-1 positive plasma and unbinding with HIV-1 negative plasma. Compared with linear HIV-1 protein sequence and HIV-1 protein structure files, only 12 clones were possible linear mimotopes of NAbs. In addition, the C40 clone located in gp41 CHR was found to be a neutralizing epitope, which could inhibit pooled HIV-1 positive plasma reaction. ConclusionBiopanning of serum IgG can yield mimotopes of HIV-1-related antigen epitopes. This methodology provides a basis for exploration into HIV-1-related antigen-antibody interactions and furthers NAb immunotherapy and vaccine design.

Functional Dissection of the Epidermal Growth Factor Receptor Epitopes Targeted by Panitumumab and Cetuximab

Cetuximab and panitumumab, two antibodies targeting the extracellular domain of the epidermal growth factor receptor (EGFR), are of major clinical importance particularly in the treatment of metastatic colorectal cancer. As patients may acquire resistance-mediating mutations within the extracellular EGFR domain, functional dissection of the exact binding sites of EGFR targeting antibodies may help predict treatment responses. We therefore assessed the epitope recognition of panitumumab by screening phage-displayed random cyclic 7mer and linear 12mer peptide libraries on this antibody. Phage screenings revealed two strong, potentially epitope-mimicking consensus motifs targeted by panitumumab. A computational approach was used to map the sequences back to the potential epitope region on domain III of EGFR. The presumed epitope regions (386)WPEXRT(391) and a biochemically similar though discontinuous region P349-F352-D355 on a neighboring loop of domain III could be confirmed as part of the functionally relevant binding site of panitumumab by site-directed mutational analysis. To more accurately differentiate the panitumumab epitope from the previously characterized cetuximab epitope, binding studies were performed on a broad range of additional mutants. Taken together, this analysis revealed two large, partially overlapping functional epitopes consisting of 17 critical amino acid positions. Four of these positions were selectively targeted by cetuximab (I467, S468, Q408, and H409), whereas another four were selectively recognized by panitumumab (W386, E388, R390, and T391). In view of the clinical significance of extracellular domain mutations, our data may help guide treatment decisions in selected patients receiving EGFR-targeted therapies.

Phage display identifies an Eastern equine encephalitis virus glycoprotein E2-specific B cell epitope

The present study identified a linear B-cell epitope in the Eastern equine encephalitis virus (EEEV) E2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an EEEV E2 specific monoclonal antibody (mAb) 7C11 and defined L/F-E/R-Y-T-W-G/R-N-H/W-P as the consensus binding motif. A sequence (321EGLEYTWGNHPP332) encompassing this consensus motif was found in the EEEV E2 glycoprotein and synthesized for further epitope confirmation. Meanwhile, the corresponding epitope peptides in E2 protein of associated alphaviruses were synthesized for specificity identification. Results showed the mAb 7C11 and murine antisera all reacted strongly against the synthesized polypeptide of EEEV antigen complex, but no reaction with Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) was detected. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against EEEV.

IgE epitopes of intact and digested Ara h 1: A comparative study in humans and rats

BackgroundAllergen epitope characterization provides valuable information useful for the understanding of proteins as food allergens. It is believed that IgE epitopes in general are conformational, nevertheless, for food allergens known to sensitize through the gastrointestinal tract linear epitopes have been suggested to be of great importance. ObjectiveThe aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. MethodsSera from five peanut allergic patients and five Brown Norway rats were used to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule to mimic epitopes using a computer-based algorithm. ResultsPatients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which by far accounted for most of the eluted peptide sequences. Epitope patterns were rather similar for both intact and digested Ara h 1 as well as for humans and rats. ConclusionsIndividual patient specific epitope patterns have been identified for the major allergen Ara h 1. IgE binding epitopes have been suggested as biomarkers for persistency and severity of food allergy, wherefore recognition of particular epitope patterns or motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergy.

Diversity of epitope recognition of IgG4 antibodies induced by birch-pollen specific immunotherapy (125.3)

Abstract Birch pollen (BP)-allergic patients sensitized to the major BP allergen, Bet v 1, often show IgE-cross-reactivity with Mal d 1 in apple and Cor a 1 in hazelnut. We recently found that specific immunotherapy (SIT) with BP induces high titers of Bet v 1-specific IgG4. In parallel, many BP-SIT-treated patients developed food-reactive IgG4. However, around 25% of patients showed no detectable food-specific IgG4 response. The diversity of epitope recognition of IgG4 antibodies (abs) in BP-SIT-treated patients developing or lacking food-reactive IgG4 were compared. Sera containing Bet v 1-, Mal d 1- and Cor a 1-specific IgE and Bet v 1-specific IgG4 with and without food-reactive IgG4 abs were included. Blocking capacity of the sera was analysed in facilitated antigen-binding and basophil activation assays. Potential epitopes recognized by allergen-specific IgE and IgG4 abs were screened by competitive immunoscreening with a random phage-displayed 12-mer peptide library. Patients showing food reactive-IgG4 with blocking capacity recognized a higher number of putative epitopes distributed over the surface of allergens. In contrast, a restricted number of putative epitopes was recognized by sera from patients who had failed to develop food-reactive IgG4. The induction of polyclonal IgG4 responses during BP-SIT may be a prerequisite for the development of abs that cross-react with Bet v 1-related food allergens and consequently, block food-reactive IgE abs.