Human Phage Display Library Research Articles

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease.

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

A single-chain Fv fragment 2A3 specific for native lysozyme: isolation from a human synthetic phage display antibody library and characterization.

We have isolated from a human synthetic phage display library a clone, 2A3, which discriminates native lysozyme from denatured forms. Binding of single-chain Fv fragments (scFvs) of the clone to native hen egg white lysozyme was competitively inhibited by native hen egg white (hew) and human (h) lysozymes. Dot blotting analysis indicated that scFv of the clone did not react with denatured lysozymes. The K(d) values for scFv of 2A3 binding to native hew- and h-lysozymes were 3.78 x 10(-9) and 9.31 x 10(-9) M, respectively, indicating that 2A3 binds more strongly to native hew-lysozyme than to native h-lysozyme. The deduced amino acid sequence of the V(H) chain-CDR3 region of 2A3 was RRYALDY, of which the Arg residues at positions 1 and 2 of the CDR3 region were observed to be extremely rare in other antibodies by homology analysis. Based on these observations, site-directed mutagenesis of the RRYALDY-coding region was carried out. The results, combined with biomolecular analyses, demonstrated that Arg residues at positions 1 and 2 of this region were important for native lysozyme-binding.

Recombinant Human Antibodies Specific for the Pfs48/45 Protein of the Malaria Parasite Plasmodium falciparum

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC50 value of 4 μM. It was found to cross react with three other microcystin variants – microcystin-RR, microcystin-LW and microcystin-LF – and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCV-positive patient.

Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control. Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value. To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease. This library was screened against a recombinant core antigen [amino acids (aa) 1-119] produced in E. coli. Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized. Both rFabC3 and rFabC14 recognize aa 1-48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C(2-20) (aa 1-20), at much lower concentrations than rFabC3. In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library. A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14. The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides. However, we confirmed the specificity of these peptides by competition experiments with sFabC14.

Characterization of anti-mouse FcγRII single-chain Fv fragments derived from human phage display libraries

Background: Few antibodies are available to study the function of the FcγRII murine immunoglobulin receptor. Human phage display libraries represent a potential source of single-chain Fv (sFv) to facilitate the study of the FcγRII murine immunoglobulin receptor. Objectives: To isolate human sFv specific for mouse FcγRII. Study design: Two human phage display libraries were selected for reactivity to mouse FcγRII. Those human anti-mouse FcγRII sFv that were derived from the libraries were characterized with respect to kinetics, cellular binding, epitope specificity and amino acid sequence. Results: Nine anti-mouse FcγRII sFv molecules were isolated from two human phage display libraries (Marks et al., J Mol Biol 1991;222:581–597; Sheets et al., Proc Natl Acad Sci USA, in press). Surface plasmon resonance (SPR) analysis revealed that the human anti-mouse FcγRII sFv had off-rates ranging from 10−2 to 10−3 s−1, with KD values calculated to range between 10−7 and 10−9 M. The binding of the FITC-labeled human anti-mouse FcγRII sFv to mouse peritoneal neutrophils was not detected by flow cytometry, due to the rapid off-rates of these monomeric proteins. However, when the human anti-mouse FcγRII sFv were coated on yellow-green latex particles, all of the human sFv were found to specifically bind to mouse peritoneal neutrophils. Deglycosylation of mouse FcγRII did not diminish the binding of these sFv, suggesting that the sFv molecules recognize a polypeptide epitope on murine FcγRII. In contrast, denaturation of mouse FcγRII dramatically reduced the binding of the human sFv, suggesting that the epitopes are conformational. Sequence analysis of the human anti-mouse FcγRII sFv revealed a high degree of structural similarity among the nine sFv. The DP73 VH gene segment was utilized by four of the nine sFv, while seven of the nine sFv contained the DPL16 Vλ gene segment. The sequence similarities between these sFv suggested that several of the human sFv may recognize a common epitope on mouse FcγRII. Epitope mapping studies demonstrated that eight of the nine human anti-mouse FcγRII sFv recognized overlapping epitopes. All of these human anti-mouse FcγRII sFv competed with the 2.4G2 rat monoclonal anti-mouse FcγRII/III antibody for binding with mouse FcγRII, suggesting that the targeted epitopes reside in or near the Fc binding pocket of mouse FcγRII. Conclusions: The availability of novel sFv recognizing mouse FcγRII will facilitate the study of receptor triggering events. Such sFv may prove useful to engage murine FcγRII for targeted cytotoxicity or immunization strategies.