Pepitope: epitope mapping from affinity-selected peptides

Identifying the epitope to which an antibody binds is central for many immunological applications such as drug design and vaccine development. The Pepitope server is a web-based tool that aims at predicting discontinuous epitopes based on a set of peptides that were affinity-selected against a monoclonal antibody of interest. The server implements three different algorithms for epitope mapping: PepSurf, Mapitope, and a combination of the two. The rationale behind these algorithms is that the set of peptides mimics the genuine epitope in terms of physicochemical properties and spatial organization. When the three-dimensional (3D) structure of the antigen is known, the information in these peptides can be used to computationally infer the corresponding epitope. A user-friendly web interface and a graphical tool that allows viewing the predicted epitopes were developed. Pepitope can also be applied for inferring other types of protein-protein interactions beyond the immunological context, and as a general tool for aligning linear sequences to a 3D structure. AVAILABILITY: http://pepitope.tau.ac.il/     

Exploring antibody recognition of sequence space through random-sequence peptide microarrays

A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes. Although peptide microarrays are physically constrained to ～10(4) peptides per array, compared with 10(8) permitted in library panning approaches such as phage display, they enable a much more high though put and direct measure of binding. Long (20 mer) random sequence peptides were chosen for this study to look at an unbiased sampling of sequence space. This sampling of sequence space is sparse, as an exact epitope sequence is unlikely to appear. Commercial monoclonal antibodies with known linear epitopes or polyclonal antibodies raised against engineered 20-mer peptides were used to evaluate this array as an epitope mapping platform. Remarkably, peptides with the most sequence similarity to known epitopes were only slightly more likely to be recognized by the antibody than other random peptides. We explored the ability of two methods singly and in combination to predict the actual epitope from the random sequence peptides bound. Though the epitopes were not directly evident, subtle motifs were found among the top binding peptides for each antibody. These motifs did have some predictive ability in searching for the known epitopes among a set of decoy sequences. The second approach using a windowing alignment strategy, was able to score known epitopes of monoclonal antibodies well within the test dataset, but did not perform as well on polyclonals. Random peptide microarrays of even limited diversity may serve as a useful tool to prioritize candidates for epitope mapping or antigen identification.     

Mimotopes to Cetuximab Isolated by mRNA-Display using Random Peptide Libraries and their Characteristics

To obtain epitope mimic peptides, mimotopes, for vaccine development, mRNA display technology was applied to Cetuximab. After six cycles of affinity selection, four clones that bound to Cetuximab were isolated that showed specific binding to the antibody in pull-down assays. Their random peptide portions were chemically synthesized and conjugated with keyhole limpet hemocyanin (KLH). Their ability to elicit production of antibodies that bind to the antibody’s original antigen, the human EGF receptor, was examined by vaccination in rabbits five times bi-weekly. Although the peptides and their KLH conjugates showed different characteristics in vitro and further analysis is needed, the results of immunizations showed that the mimotope peptides isolated from mRNA display libraries could generate antibodies against the antibody’s original antigen in vivo.     

By-passing selection: direct screening for antibody–antigen interactions using protein arrays

We have developed a system to identify highly specific antibody–antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody–antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this ‘naive’ screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

Different immune response of mice immunized with conjugates containing multiple copies of either consensus or mixotope versions of the V3 loop peptide from human immunodeficiency virus type 1

The critical role that antibody responses to the V3 loop epitope play in human immunodeficiency virus type 1 (HIV-1) neutralization has caused this peptide to be used in many HIV-1 vaccine candidates. To enhance cross-reactivity toward several V3 sequences, a database of 50 peptides of the V3 region from HIV-1 subtype A was used to design both a consensus peptide and a combinatorial peptide (mixotope) library representative of these sequences. The two immunogens (consensus and mixotope) were incorporated into multiple antigen peptide (MAP) constructions, conjugated to a recombinant surface antigen from hepatitis B virus (HbsAg) carrier protein, and inoculated to mice in combination with a C4 (CD4-binding) peptide MAP construction, also conjugated to HBsAg. The respective responses and cross-reactivity to several V3 loop sequences of both types of immunogens were compared. Mice inoculated with the V3 consensus-MAP-HBsAg + C4-MAP-HBsAg mixture elicited higher antibody responses than those given the V3 mixotope-MAP-HBsAg + C4-MAP-HBsAg mixture. In addition, pooled serum from the first group of immunogens analyzed at dilution 1:100 had higher cross-reactivity against V3 peptides on cellulose membranes than those from mice given the combinatorial immunogen. Fine epitope mapping of both consensus and C4 peptide by the spot synthesis technique showed that sera of the first group strongly recognized both sequences in their entirety, whereas mice immunized with the mixotope library recognized only the N-terminal region of V3. These results seem to suggest that the V3 consensus peptide is superior to the combinatorial strategy in inducing potent and cross-reactive responses to HIV.     

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100_209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the _mutgp100_201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the _mutgp100_209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8⁺ T cell stimulation in vitro similar to the _wtgp100_209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8⁺ T cell response also towards N-terminally extended versions of the minimal epitope.     

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5 terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.     

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Background: Development of functional monoclonal antibodies against intractable GPCR targets.Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large ‘family A’ of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor.The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.     

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands

cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody–antigen, protein– protein and DNA–protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.     

Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.     

Mimotopes for Alloreactive and Conventional T Cells in a Peptide–MHC Display Library

The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins.     

Molecular basis for antagonistic activity of anifrolumab,an anti-interferon–α receptor 1 antibody

Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon α receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R²⁷⁹. Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope. © 1998 John Wiley & Sons, Ltd.     

A novel method for construction of gene fragment library to searching epitopes

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.     

Epitope Identification from Fixed-complexity Random-sequence Peptide Microarrays

Antibodies play an important role in modern science and medicine. They are essential in many biological assays and have emerged as an important class of therapeutics. Unfortunately, current methods for mapping antibody epitopes require costly synthesis or enrichment steps, and no low-cost universal platform exists. In order to address this, we tested a random-sequence peptide microarray consisting of over 330,000 unique peptide sequences sampling 83% of all possible tetramers and 27% of pentamers. It is a single, unbiased platform that can be used in many different types of tests, it does not rely on informatic selection of peptides for a particular proteome, and it does not require iterative rounds of selection.In order to optimize the platform, we developed an algorithm that considers the significance of k-length peptide subsequences (k-mers) within selected peptides that come from the microarray. We tested eight monoclonal antibodies and seven infectious disease cohorts. The method correctly identified five of the eight monoclonal epitopes and identified both reported and unreported epitope candidates in the infectious disease cohorts. This algorithm could greatly enhance the utility of random-sequence peptide microarrays by enabling rapid epitope mapping and antigen identification.Antibodies play a central role in the immune system and in modern health care and medical research. They are commonly used as affinity reagents in research and diagnostic applications and have emerged as an important class of therapeutics (1). When new affinity reagents are being generated, it is useful to know the target sequence (epitope) bound by the antibody in question. Many methods have been developed to accomplish this, including peptide tiling and phage, bacteria, and mRNA display (2–4). Especially for newly discovered diseases, such as Middle East respiratory syndrome (5), knowing the epitope(s) that elicits a humoral response enables the production of diagnostics and vaccines. Large-scale mapping of cohorts infected with the same disease may guide the development of universal vaccines for flu and other infections. Crystal structure and B-cell sequencing provide the most detailed information about antibody targeting, but in practice these are cost prohibitive and rarely done. Library-panning-type approaches use bacteria or phages to display peptide sequences, avoiding costly crystallization or synthesis steps, and are common approaches for linear epitope mapping (3, 6). Recently, bacterial display methods have been used to discover antigens in celiac disease (2). Tools for probing the “memory” of the immune system could reveal a wealth of information about an individual''s health status and antibody repertoire. Although display techniques are effective and result in highly accurate and specific linear epitope determination (7, 8), they have hidden and poorly understood biases regarding sequence populations (9–11) and rely on selection steps that eliminate certain sequences in favor of others. This creates issues with cost and reliability at scale, and information is discarded as the selection process becomes increasingly stringent. As a rapid identification method, panning is not optimal.Peptide array technologies provide an alternative approach. They are simple and reproducible, they provide information about binders and non-binders, and they can be low cost if mass produced, but they represent a smaller sequence library than phage display and contain only linear sequences. This might seem like a disadvantage, but in practice, linear epitopes are actually quite common in nature, and even mimotopes can provide useful, if indirect, information about non-linear epitopes. Microarrays containing hundreds of thousands of peptides are becoming more accessible, reducing the impact of smaller libraries. Additionally, microarrays are capable of displaying interactions between antibodies and peptides with short, gapped sequences containing four to six anchor residues, which seem to cover a sizable class of antibodies (12, 13).To date the most common approach to designing peptide microarrays has been to tile sequences from a known protein or proteome of interest and find sequences that bind the target (4, 14–17). Recently this technique has been scaled to whole proteomes using arrays containing millions of sequences (14, 16). This approach is effective on a single-protein scale, but problems arise when one is looking for specific epitope sequences in the presence of millions of other peptides. Cross-reactivity of antibodies to non-target peptides often obscures the eliciting antigen (14). This might be due in part to the fact that tiled peptides are fundamentally different from folded proteins, and inaccessible parts of a protein are likely to be exposed when linear pieces of it are tiled. Additionally, there are many common n-mers across apparently unrelated pathogens. It might be possible to address this problem using motif-based discovery rather than peptide-based discovery. Short motifs (4- to 5-mers) will likely appear multiple times in a given peptide library. Longer sequences (6- to 12-mers) should appear more rarely. We propose that a platform for epitope discovery should focus on representing as many unique short motifs as possible, rather than providing longer, overlapping sequences from a particular set of proteins.Previously our group used random-sequence peptide microarrays to diagnose disease using immunosignatures (18, 19). The immunosignaturing effect relies on the interaction of serum antibodies with random-sequence peptides bound to a microarray. When properly trained on well-validated cohorts, this indirect information provides very discerning and predictive information about disease states in blinded individuals (18, 20–23). Although immunosignatures are sensitive and specific as a diagnostic tool, a link has not been established between immunosignature profiles and actual sequences of signature peptides. This was attempted in a previous study by our group in which we evaluated an array of 10,000 17-mer peptides as a platform for epitope mapping. Although useful for predicting linear sequences for some monoclonal antibodies, it offered virtually no predictive power in serum samples from mice immunized to a known antigen (24). Since then, advances in in situ synthesis techniques have enabled our group to produce microarrays containing several million peptides per slide (25). These arrays contain >27% of possible pentamers and 83% of possible tetramers. Although it lacks the majority of pentamers, this is a fairly dense sampling of short peptide sequences that might be useful for epitope mapping.Here we report on a general approach that uses random sequence peptide arrays to map epitopes. We demonstrated this by identifying epitope sequences from a set of monoclonal antibodies. We then used the same technique with different disease cohorts containing antibodies of unknown specificity, revealing both previously discovered and new epitopes. The study described here is the first attempt at deciphering a microarray with fixed but random peptide sequences for epitopes that does not a priori assume a set of eliciting proteins.     

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide–beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-β-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μκ) of two murine B-cell lymphoma cell lines (antigen unknown).     

B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 × 10⁸ clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant K_d = 1 × 10^–7 M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.