Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents

Structural insights into AChE inhibition by monoclonal antibodies

International audienceThe target sites of three inhibitory monoclonal antibodies, Elec403, 408 and 410, on eel AChE have been defined previously. Elec403 and 410 are directed toward distinct but overlapping epitopes at the enzyme peripheral site, while Elec408 binds to a distinct regulatory site on the enzyme surface, where the "back door" may be located. Elec410 also inhibits Bunganus fasciatus AChE. To investigate the molecular determinants for AChE inhibition by these antibodies, we have cloned and sequenced the IgGs, generated, purified, characterized the Fab molecules, and initiated crystallographic and theoretical modeling studies. Preliminary data are presented

Molecular characterization of monoclonal antibodies that inhibit acetylcholinesterase by targeting the peripheral site and backdoor region.

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis

Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases

International audienc

Physical and functional characterization of the purified Fabs (typical experiments).

<p>(<b>A</b>) MALDI-TOF mass spectrometry profiles, showing both the di-charged and mono-charged entities. Note the satisfactory homogeneity in mass of the latter. (<b>B</b>) Electrophoresis patterns obtained by SDS-PAGE <i>in </i><i>non-reducing </i><i>conditions</i> (12.5% PhastGel, left) and native-PAGE (7.5% PhastGel, center) with migration from the cathode (top) toward the anode (bottom), and by isoelectric focusing (pI 3-9 PhastGel, right). The three Fabs are more homogenous in mass than in charge, a feature that likely result from variations in the C-terminus generated by papaine cleavage of the CH chain; yet, all isoforms bind EeAChE equally, as verified by a native-PAGE mobility shift assay (not shown). The neutral average pI of Fab408 and cationic average pIs of Fab403 and Fab410 are evident. (<b>C</b>) Inhibition of native EeAChE (closed symbols, full lines) and N-deglycosylated EeAChE (open symbols, dashed lines) by the three Fabs. The higher residual activity recorded at saturating concentration of Fab408, compared with Fab403 and Fab410, is evident. Removal of the six N-linked glycan chains of EeAChE results in 6.5-fold higher affinity for Fab403 but unaltered residual activity at saturating Fab403 concentration. Data points correspond to the average values of duplicates or mean values of triplicates. Non-linear fitting of the data points used a sigmoidal equation. Mean values of the mass, pI, IC50 and residual activity of each Fab are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077226#pone-0077226-t001" target="_blank">Table 1</a>.</p

Structure of Fab408 and homology models of Fab403 and Fab410.

<p><b>Left</b>: Overall views of (<b>A</b>) Fab408, with a light blue L chain and yellow H chain; (<b>B</b>) Fab403, with a violet L chain and orange H chain; (<b>C</b>) Fab410, with a dark blue L chain and salmon H chain, displayed with their N-terminal variable region on top and C-terminal constant region at bottom. CDR-L1, CDR-L2, CDR-L3 are displayed in blue, light green, dark green and CDR-H1, CDR-H2, CDR-H3 in red, orange, purple. The extended CDR-H3 in Fab408 is clearly visible. <b>Center</b>: Close-up views of the combining sites, displayed as molecular surfaces with the CDRs colored as on the left panels and labeled. Differences in the sizes of the CDRs and the shapes of the combining surfaces (pocket in Fab408 <i>versus</i> extended surfaces in Fab403 and Fab410) are evident. <b>Right</b>: Distribution of the electrostatic potentials mapped on the Fab molecular surfaces at -3 kT/e (red) to +3 kT/e (blue) (same orientation as on the central panels). Note the electronegative combining site in Fab408 <i>versus</i> the electropositive combining sites in Fab403 (centered around the L-chain CDRs) and Fab410 (centered around the H-chain CDRs).</p

Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase–deficient (Tk−) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk− rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins

Sequences and numbering of the three anti-EeAChE Fabs.

<p>The light (L, top) and heavy (H, bottom) chains are displayed. The residue numbering and secondary structure elements displayed above the alignment are those of Fab408. Conserved residues are shown on a <i>black</i> background and non-conserved residues on a <i>white</i> background. The CDRs, defined according to <a href="http://www.bioinf.org.uk/abs/" target="_blank"><u>http://www.bioinf.org.uk/abs/</u></a> (cf. Figure S1 in File S1), are highlighted as grey boxes and labeled.</p

Differential biopanning strategy to select conformational mimotopes of desired specificity.

<p>HIV gp160 was bound to ELISA plates under native or denaturing conditions. After incubation with serum of monkey RKl-8 (which was chronically infected with a clade C simian-human immunodeficiency virus (SHIV) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038943#pone.0038943-Humbert1" target="_blank">[25]</a> and had high titers of anti-HIV nAbs) followed by washing, Abs were eluted. Abs eluted from the native protein antigen (“conformational” Abs) were used for the positive selection rounds; Abs eluted from the denatured protein (“linear” Abs) were used for the negative rounds. A total of three biopanning rounds were performed and mimotopes selected by this strategy were tested for specificity and conformation dependence with rhesus monkey sera.</p

Strategy of isolation of epitope-specific Abs.

<p>After differential biopanning of phage display peptide library, mimotopes representing conformational epitopes are sequenced, and the inserts plus M13 flanking sequences are cloned into a bacterial expression vector encoding fluorescent protein. After purification of the resulting mimotope fusion proteins, binding assays to assure maintenance of the correct three-dimensional structure of mimotope insert are conducted. This is followed by single-cell sorting for specific IgG-positive memory B cells, single-cell RT-PCR and cloning of RM V_L and V_H regions into pFUSE2-type vectors encoding the backbone of human IgG1.</p