3-O-sulfated oligosaccharide structures are recognized by anti-heparan sulfate antibody HS4C3

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O- and 6-O-sulfated hexa- to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mm. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mm salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the anti-thrombin binding pentasaccharide sequence.     

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.     

A review: Cloning of human antibodies by phage display

Antibodies play a pivotal role in human health and disease. The application of phage display technology represents another milestone in the attempt to gain a better understanding of human antibodies. Immunoglobulin phage display permits human monoclonal antibodies for the first time to be readily available for analysis and for therapeutic use. Recent developments in molecular biology, in particular the polymerase chain reaction, have made it possible to amplify, clone, and express human antibody fragments in prokaryotic organisms. Phagemid display vectors have a distinct advantage over conventional cell culture technology used to immortalize human antibodies, in that one may quickly survey huge immunoglobulin repertoires for an antibody of desired specificity. Dual expression of immunoglobulin variable region light and heavy chain fragments permits combinatorial shuffling and thus an increase in diversity.The development of sophisticated computer algorithms, such as LINUS,⁵⁷ that can predict the three-dimensional structure of proteins from DNA sequences will have an enormous influence on the characterization and design of human antibodies. Future advances in computer software will be needed to aid in the identification of unique antibody sequence motifs expressed during disease and in the design of antibodies with defined functional epitopes.     

Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein

Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) gamma1/kappa antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, K(d)) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.     

Selection and characterization of human monoclonal antibodies against Abrin by phage display

Abrin is a highly potent and lethal type II ribosome inactivating toxin that may be used as a biological warfare agent. To date, no human anti-Abrin antibodies have yet to be reported. Herein, we describe the selection and characterization of two human monoclonal antibodies, termed E12 and RF12, which are capable of binding native Abrin with high affinity and specificity. Through surface plasmon resonance studies, we have determined the association and dissociation rate constants and the cross-reactivity for both antibodies. In our developed Biacore-based Abrin detection system, the limit of detection of antibodies E12 and RF12 is 35 and 75 ng/mL, respectively. These concentrations are about 5 x 10(4)-fold lower than the extrapolated Abrin human LD(50). In sum, our data demonstrated the power of human antibody phage display libraries and the promise of these antibodies as detection devices for Abrin.     

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.     

T4 bacteriophage as a phage display platform

Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.     

Human anti-self antibodies with high specificity from phage display libraries.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.     

Localization of melanin in conidia of Alternaria alternata using phage display antibodies

Melanins derived from 1,8-dihydroxynaphthalene (DHN) are important for the pathogenicity and survival of fungi causing disease in both plants and animals. However, precise information on their location within fungal cell walls is lacking. To obtain antibodies for the immunocytochemical localization of melanin, 83 phage antibodies binding to 1,8-DHN were selected from a naive semisynthetic single-chain Fv (scFv) phage display library. Sequence analysis of the heavy chain binding domains of 17 antibodies showed a high frequency of positively charged amino acids. One antibody, designated M1, was characterized in detail. M1 bound specifically to 1,8-DHN in competitive inhibition enzyme-linked immunosorbent assays, showing no cross-reaction with nine structurally related phenolic compounds. Epitope recognition required two hydroxyl groups in a 1,8 configuration. M1 also bound to naturally occurring melanin isolated from mycelia of Alternaria alternata, suggesting that epitopes remain accessible in polymerized melanin. Transmission electron microscopy-immunogold labeling, using M1 in the form of soluble scFv fragments, showed that melanin was located in the septa and outer (primary) walls of wild-type A. alternata conidia, but not those of an albino mutant, AKT88-1. The M1 antibody provides a new tool for detecting melanized pathogens in plant and animal tissues and for precisely mapping the distribution of the polymer within spores, appressoria, and hyphae.     

Affinity improvement of the high-affinity immunoglobulin E receptor by phage display

The immunoglobulin E (IgE)-binding site of its high-affinity receptor is localized in the second immunoglobulin-like domain (D2) of the alpha-subunit (Fc epsilon RI alpha). In this study, the randomized pentapeptides were introduced between Glu(132) and Ile(138) of Fc epsilon RI alpha D2 and displayed on a filamentous phage. After eight rounds of panning, a phage clone having a mutation of Asp(135)Tyr(136)Met(137) in Fc epsilon RI alpha D2 was obtained. The binding affinity of the mutant phages to immobilized IgE was approximately 500 times higher than that of the wild type. The mutant phages competitively inhibited the binding of IgE to the soluble receptor at a 50% inhibition (IC(50)) value of 116 pM. The mutant Fc epsilon RI alpha D2, which had been expressed as a fusion protein with glutathione S-transferase in Escherichia coli, also showed higher IgE-binding capacity than the wild type. The mutant Fc epsilon RI alpha D2 is expected to manifest its improved IgE-binding affinity together with any fusion partner.     

Characterization of prostate-specific antigen binding peptides selected by phage display technology

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.     

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.     

Using phage display to select antibodies recognizing post-translational modifications independently of sequence context

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.     

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.     

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     

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1 + HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC₅₀ values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40 μM and 2.20 μM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K_d value of 3.09 × 10⁻¹¹ M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.     

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.

Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.     

一种从抗狂犬病病毒噬菌体抗体库中筛选和验证中和抗体的方法

目的建立一种从噬菌体库抗体库中高效筛选和验证抗狂犬病病毒中和抗体的方法。方法①定性分析:从经过灭活的CVS-11筛选噬菌体抗体库中挑取单克隆于96孔板中培养,制备噬菌体抗体,取培养上清进行快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT),选择可明显抑制病毒感染的克隆测序,获得具有中和活性的抗体可变区序列;②定量分析:挑选有中和活性的克隆,重新制备噬菌体抗体颗粒,纯化后进行RFFIT分析;扩增抗体可变区基因,构建真核瞬时表达质粒。瞬转HEK-293 EBNA1细胞,培养上清经Mabselect SuRe亲和纯化后测定抗体比活。结果定性分析获得的噬菌体抗体颗粒与全分子抗体体外中和活性显著相关;剔除低活性序列后,活性高于0.5 IU/mL的噬菌体抗体颗粒与其全分子抗体体外中和活性之间无显著相关;所有纯化后噬菌体抗体颗粒活性>0.5 IU/mL的序列其全分子抗体体外中和活性均>500 IU/mg。结论构建了一种从抗狂犬病病毒噬菌体库中高效筛选、验证中和抗体的方法。