Oxidoreductase activity of multifunctional monoclonal antibody B13-DE1

The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 μmol/l and 0.0056 s(-1) , respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical.     

Three‐dimensional structure,binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

Unlike other known anti‐fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (K_D ～ 70 pM) and a fast association rate (k_on ～ 2 × 10⁷ M^?1 s^?1). The three‐dimensional structure of the Fab 43.1—fluorescein complex was resolved at 2.4 Å resolution. The antibody binding site is exclusively assembled by the CDR loops. It is comprised of a 14 Å groove‐shaped entrance leading to a 9 Å by 7 Å binding pocket. The highly polar binding pocket complementary encloses the fluorescein's carboxyphenyl moiety and tightly fixes it by multiple hydrogen bonds. The fluorescein's xanthene ring is embedded in the more hydrophobic groove and stacked between the side chains of Tyr37_L and of Arg99_H providing conditions for an excited state electron transfer process. In comparison to fluorescein, the absorption spectrum of the complex in the visible region is shifted to the “red” by 23 nm. The complex demonstrates a very weak fluorescence (Φ_c = 0.0018) with two short lifetime components: 0.03 ns (47%) and 0.8 ns (24%), which reflects a 99.8% fluorescein emission quenching effect upon complex formation. The antibody 43.1 binds fluorescein with remarkable affinity, fast association rate, and strongly quenches its emission. Therefore, it may present a practical interest in applications such as molecular sensors and switches. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 234–243, 2016.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Hydrophobic interactions are the driving force for the binding of peptide mimotopes and <Emphasis Type="Italic">Staphylococcal </Emphasis>protein A to recombinant human IgG1

We studied the interaction of several nona-peptide mimotopes of different sequence and Staphylococcal protein A (SpA) with a recombinant human IgG1 antibody using isothermal titration calorimetry (ITC). The amino acid primary structure of the peptides was varied in order to identify the specific antibody-peptide binding sites. Additionally, the influence of temperature and salt concentration was investigated. An attempt was made to elucidate the structural changes upon complex formation using the determined thermodynamic parameters. The amino acid composition of the mimotopes determined their binding affinity. The binding constant K _a of the mimotopes was in the range 1 × 10⁴ to 1 × 10⁶ M⁻¹. The binding constant of SpA was on the average about three orders of magnitude higher than that of the peptides. The binding constant of the peptides and of SpA decreased with temperature and the binding process was connected with negative changes in enthalpy, entropy, and heat capacity. The binding of the mimotopes to the Fab part of the IgG1 antibody and binding of SpA to the Fc part of the IgG1 antibody were mainly driven by hydrophobic effects and associated with a relatively large change in water-accessible surface area. Determinants for a strong/reduced antibody-peptide binding were identified.     

Isolation of B subunit‐specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2

Shiga toxin 2 (Stx2)‐specific mAb‐producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)‐specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat‐labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)‐specific whereas five were Stx2B‐specific antibody‐producing clones. The in vitro neutralization activity of Stx2B‐specific mAbs against Stx2 was greater than that of Stx2A‐specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B‐specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B‐specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V_H and V_L regions of clones 45 and 75D9 were determined. Our Stx2B‐specific mAbs may be new candidates for the development of mouse‐human chimeric Stx2‐neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.     

Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1.

A systematic exploration of the V(H)2/V(kappa)12-13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

A naturally occurring αs1‐casein‐derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum‐free conditions

Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra‐filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α_s1‐CN (f16‐23) (RPKHPIKH), α_s1‐CN (f16‐24) (RPKHPIKHQ), α_s1‐CN (f17‐25) (PKHPIKHQG), α_s1‐CN (f46‐52) (VFGKEKV), α_s1‐CN (f94‐105) (HIQKEDVPSER), and β‐CN (f121‐128) (HKEMPFPK), were identified. One of the major peptides, the N‐terminal fragment of α_s1‐casein, varied structurally during early lactation: α_s1‐CN (f17‐25) (PKHPIKHQG) and α_s1‐CN (f16‐23) (RPKHPIKH)/α_s1‐CN (f16‐24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α_s1‐CN (f16‐23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum‐free conditions in a dose‐dependent manner, in consequence of caspase‐3 and caspase‐9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti‐apoptotic agent in cell culture medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Polishing approach with fully connected flow‐through purification for therapeutic monoclonal antibody

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi‐continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow‐through polishing technologies that include activated carbon, cation exchange, and anion‐exchange chromatography. In this report, we evaluated fully‐connected pool‐less polishing with three flow‐through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory‐scale pool‐less processing was achieved without utilizing in‐line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow‐through configurations of polishing steps were evaluated: a two‐step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory‐scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural basis for preferential binding of non-ortho-substituted polychlorinated biphenyls by the monoclonal antibody S2B1

Polychlorinated biphenyls (PCBs) are a family of 209 isomers (congeners) with a wide range of toxic effects. In structural terms, they are of two types: those with and those without chlorines at the ortho positions (2, 2', 6 and 6'). Only 20 congeners have no ortho chlorines. Three of these are bound by the aryl hydrocarbon receptor and are one to four orders of magnitude more toxic than all others. A monoclonal antibody, S2B1, and its recombinant Fab have high selectivity and nanomolar binding affinities for two of the most toxic non-ortho-chlorinated PCBs, 3,4,3',4'-tetrachlorobiphenyl and 3,4,3',4',5'-pentachlorobiphenyl. To investigate the basis for these properties, we built a three-dimensional structure model of the S2B1 variable fragment (Fv) based on the high-resolution crystallographic structures of antibodies 48G7 and N1G9. Two plausible conformations for the complementarity-determining region (CDR) H3 loop led to two putative PCB-binding pockets with very different shapes (models A and B). Docking studies using molecular mechanics and potentials of mean force (PMF) indicated that model B was most consistent with the selectivity observed for S2B1 in competition ELISAs. The binding site in model B had a deep, narrow pocket between V(L) and V(H), with a slight constriction at the top that opened into a wider pocket between CDRs H1 and H3 on the antibody surface. This binding site resembles those of esterolytic antibodies that bind haptens with phenyl rings. One phenyl ring of the PCB fits into the deep pocket, and the other ring is bound in the shallower one. The bound PCB is surrounded by the side chains of TyrL91, TyrL96 and TrpH98, and it has a pi-cation interaction with ArgL46. The tight fit of the binding pocket around the ortho positions of the bound PCBs indicates that steric hindrance of ortho chlorines in the binding site, rather than induced conformational change of the PCBs, is responsible for the selectivity of S2B1.     

Multistage aqueous two‐phase extraction of a monoclonal antibody from cell supernatant

This article presents results of continuous multistage aqueous two‐phase extraction of an immunoglobulin G1 from cell supernatant in a mixer‐settler unit. An aqueous two‐phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three‐stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:925–936, 2015     

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol–H₂O₂–horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intesity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects. © 1997 John Wiley & Sons, Ltd.     

Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3

The amino acid sequences of the heavy- and light-chain variable regions of the high-affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, gamma-carbohydrate group is solvent-exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure-activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257-3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding.     

Characteristics of monoclonal antibody binding with the <Emphasis Type="Italic">C</Emphasis> domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of complexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 ± 0.4 and 2.5 ± 0.4 nM, respectively, for recombinant tACE and 4.7 ± 0.5 and 1.6 ± 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.