Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Mapping epitopes of neutralizing monoclonal antibodies using phage random peptide libraries

Identification of protective determinants from microbial proteins is a necessary step in the rational design of subunit vaccines. We have previously used a synthetic peptide scan (Pepscan) assay to map a panel of eight neutralizing monoclonal antibodies (mAb; designated as C1.1 to C1.8) to a common motif sequence from Chlamydia trachomatis. In the present study, five of the eight mAbs were used to screen phage random peptide libraries. mAbs C1.1 and C1.3 selected a motif sequence of G-L-X-N-D from a pIII-based phage random peptide library and a motif sequence of G-X-X-N-D from a pVIII-based random peptide library while mAbs C1.6 to C1.8 failed to select recognizable motifs from either of the phage libraries. However, C1.6 to C1.8 bound to the same motif sequence displayed on phage when the appropriate conformational constraints were imposed onto the motif sequence. Thus the specificity of the mAbs identified on Pepscan assays correlates with the mAbs’ dependence on local epitope constraints displayed on the phage surface. Received 12 August 1996/ Accepted in revised form 03 November 1996     

Cyclic peptides selected by phage display mimic the natural epitope recognized by a monoclonal anti-colicin A antibody.

A 10-mer random peptide library displayed on filamentous bacteriophage was used to determine the molecular basis of the interaction between the monoclonal anti-colicin A antibody 1C11 and its cognate epitope. Previous studies established that the putative epitope recognized by 1C11 antibody is composed of amino acid residues 19-25 (RGSGPEP) of colicin A. Using the phage display technique it was confirmed that the epitope of 1C11 antibody was indeed restricted to residues 19-25 and the consensus motif RXXXPEP was identified. Shorter consensus sequences (RXXPEP, RXXEP, KXXEP) were also selected. It was also demonstrated that the disulfide bond found in one group of the selected peptides was crucial for 1C11 antibody recognition. It was shown that cyclization of the peptides by disulfide bond formation could result in a structure that mimics the natural epitope of colicin A.     

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.     

SIV envelope glycoprotein epitopes recognized by antibodies from infected or vaccinated rhesus macaques

We analyzed SIV-specific monkey sera to localize B-cell epitopes of the envelope glycoprotein of SIV (gp130), using overlapping synthetic peptides representing the entire SIV gp130 protein and sera from experimentally infected monkeys and monkeys immunized with whole, inactivated SIV. A B-cell epitope which induces neutralizing antibody production and T-cell responses was characterized as well as a new B-cell epitope and a previously described neutralizing epitopes. Vaccinated monkey sera recognize the three epitopes differentially relative to unimmunized controls, and a correlation appears to exist between degree of cross-neutralization by infected monkey sera and degree of binding to these three regions.     

从噬菌体表面展示肽库中筛选衣原体单克隆抗体识别的抗原表位

利用抗体捕获法,经三轮淘洗,从表面展示随机肽序列的噬菌体文库中筛选到与衣原体单克隆抗体C17特异结合的噬菌体克隆,其一致序列为：（L／I）PGGS（P／W）,竞争抑制实验表明含特异序列的克隆能与天然抗原竞争。据此,我们认为此序列为衣原体的B细胞抗原表位。     

交替洗脱法筛选高亲和力噬菌体肽

以人胰岛素为靶蛋白从七肽展示库中筛选高亲和力噬菌体肽,在洗脱阶段采用酸性洗脱液和高浓度靶蛋白溶液进行4次交替洗脱,选择性回收高亲和力噬菌体肽。测定滴度计算回收率,ELISA法分别测定噬菌体洗脱液整体亲和力和噬菌体单克隆的结合特性并计算亲合率。洗脱步骤采用4次交替洗脱后,第二轮第4次噬菌体的回收率比第一轮增长了1800倍,高亲和力噬菌体在洗脱液中所占比例也迅速提高,第二轮第4次洗脱液中达75％,在第三轮的各次洗脱液中几乎均达100％。建立了一种快速筛选高亲和力噬菌体肽的方法,改进后的筛选方法能使高亲和力噬菌体肽的筛选工作更为简便且效果显著。     

Schistosomajaponicum： Isolation and Identification of Peptides Mimicking Ferritin Epitopes from Phage Display Library

Although schistosomicidal drug and other control measures (including hygiene and snail control) are availableschistosomiasis continues to afflict an estimated 200 million and kill 20 thousand people every year, new approachefor controlling this disease are urgently needed. Cocktail vaccine, such as multiple antigen peptidvaccine, emerged as the most potentially powerful meansFor multiple antigen peptide vaccine that bases on domainor subdomains of antigens, the key is to obtain antigeniepito…     

鉴定单克隆抗体识别蛋白质抗原表位方法的建立

为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法,选择程序死亡受体-1(PD-1)作为目的蛋白。基于丙氨酸扫描策略,建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法,确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体,鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位,与已报道的基于晶体结构的抗原表位高度一致,表明本方法操作简单、准确性高,可用于治疗性单克隆抗体的抗原表位作图。     

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.     

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.     

Stepwise prediction of conformational discontinuous B-cell epitopes using the Mapitope algorithm

Mapping the epitope of an antibody is of great interest, since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. Here we present Mapitope, a computer algorithm for epitope mapping. The algorithm input is a set of affinity isolated peptides obtained by screening phage display peptide-libraries with the antibody of interest. The output is usually 1-3 epitope candidates on the surface of the atomic structure of the antigen. We have systematically tested the performance of Mapitope by assessing the effect of the algorithm parameters on the final prediction. Thus, we have examined the effect of the statistical threshold (ST) parameter, relating to the frequency distribution and enrichment of amino acid pairs from the isolated peptides and the D (distance) and E (exposure) parameters which relate to the physical parameters of the antigen. Two model systems were analyzed in which the antibody of interest had previously been co-crystallized with the antigen and thus the epitope is a given. The Mapitope algorithm successfully predicted the epitopes in both models. Accordingly, we formulated a stepwise paradigm for the prediction of discontinuous conformational epitopes using peptides obtained from screening phage display libraries. We applied this paradigm to successfully predict the epitope of the Trastuzumab antibody on the surface of the Her-2/neu receptor in a third model system.     

The rescue by phage display of human fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes

Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5′ primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.     

Identification of the epitopes of calcitonin gene-related peptide (CGRP) for two anti-CGRP monoclonal antibodies by 2D NMR.

The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.     

Identification of inorganic crystal-specific sequences using phage display combinatorial library of short peptides: A feasibility study

The feasibility of using a combinatorial phage display library of decapeptides to identify ligands which can interact with the surface of a crystal was assessed using geological calcium carbonate as a model. Two relatively strong binding clones were identified by ELISA, sequenced and the encoded oligopeptides were prepared by solid phase synthesis and their properties compared with those of casein hydrolysate.     

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins

Summary Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.Abbreviations HIV Human Immunodeficiency Virus - SIV Simian Immunodeficiency Virus - HTLVI Human T cell Leukaemia Virus - AIDS Acquired Immune Deficiency Syndrome - mAb Monoclonal Antibody - ELISA Enzyme Linked Immunosorbent Assay - PBS Phosphate Buffered Saline     

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage‐displayed 12‐mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero^® anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.