Search

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs., (Copyright © 2011. Published by Elsevier Inc.)

For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live-cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG-coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS). Controls include IgG-coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG-FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.

Herein we demonstrate a sensitive approach for protein detection based on peak shifts of localized surface plasmon resonance (LSPR) induced by aptamer-antigen-antibody sandwich structures. The applicability of the proposed method is demonstrated using human α-thrombin as a model analyte. While the binding of thrombin to its specific receptor, thrombin binding aptamer (TBA) modified on Au nanorods (AuNRs), causes a measurable LSPR shift, a subsequent binding of an anti-thrombin antibody to the captured thrombin can exhibit a nearly 150% amplification in the LSPR response. This enhanced signal essentially leads to an improvement of limit of detection (LOD) by more than one order of magnitude. In addition, the use of TBA as thrombin recognition units makes the biosensor reusable. The feasibility of the proposed method was further exploited by the detection of thrombin in human serum, opening the possibility of a real application for diagnostics and medical investigations., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.

We present a web interface which allows us to conveniently set up calculations based on the BioFET-SIM model. With the interface, the signal of a BioFET sensor can be calculated depending on its parameters, as well as the signal dependence on pH. As an illustration, two case studies are presented. In the first case, a generic peptide with opposite charges on both ends is inverted in orientation on a semiconducting nanowire surface leading to a corresponding change in sign of the computed sensitivity of the device. In the second case, the binding of an antibody/antigen complex on the nanowire surface is studied in terms of orientation and analyte/nanowire surface distance. We demonstrate how the BioFET-SIM web interface can aid in the understanding of experimental data and postulate alternative ways of antibody/antigen orientation on the nanowire surface.

Humanized antibodies are constructed by CDR grafting, while retaining those murine framework residues that influence the antigen-binding activity. To reduce the immunogenicity of CDR-grafted humanized antibodies, the murine content in the CDR-grafted humanized antibodies is minimized through SDR grafting. Within each CDR, there are more variable positions that are directly involved in the interaction with antigen, i.e., specificity-determining residues (SDRs), whereas there are more conserved residues that maintain the conformations of CDRs loops. SDRs may be identified from the 3D structure of the antigen-antibody complex and/or the mutational analysis of the CDRs. An SDR-grafted humanized antibody is constructed by grafting the SDRs and the residues maintaining the conformations of the CDRs onto human template, and its immunogenic potential is evaluated by measuring the reactivity to the sera from patients who had been immunized with the parental antibody.

Background: Prediction of B-cell epitopes from antigens is useful to understand the immune basis of antibody-antigen recognition, and is helpful in vaccine design and drug development. Tremendous efforts have been devoted to this long-studied problem, however, existing methods have at least two common limitations. One is that they only favor prediction of those epitopes with protrusive conformations, but show poor performance in dealing with planar epitopes. The other limit is that they predict all of the antigenic residues of an antigen as belonging to one single epitope even when multiple non-overlapping epitopes of an antigen exist., Results: In this paper, we propose to divide an antigen surface graph into subgraphs by using a Markov Clustering algorithm, and then we construct a classifier to distinguish these subgraphs as epitope or non-epitope subgraphs. This classifier is then taken to predict epitopes for a test antigen. On a big data set comprising 92 antigen-antibody PDB complexes, our method significantly outperforms the state-of-the-art epitope prediction methods, achieving 24.7% higher averaged f-score than the best existing models. In particular, our method can successfully identify those epitopes with a non-planarity which is too small to be addressed by the other models. Our method can also detect multiple epitopes whenever they exist., Conclusions: Various protrusive and planar patches at the surface of antigens can be distinguishable by using graphical models combined with unsupervised clustering and supervised learning ideas. The difficult problem of identifying multiple epitopes from an antigen can be made easied by using our subgraph approach. The outstanding residue combinations found in the supervised learning will be useful for us to form new hypothesis in future studies.

Deposition of a tapered thin film of Ta(2)O(5) onto a single-mode, polarization-insensitive slab glass waveguide enables the resultant structure to serve as a simple, inexpensive yet highly sensitive polarimetric interferometer for trace, even ultra-trace, detection of chemical and biochemical analytes. By comparing the measured refractive-index sensitivity with that simulated based on a four-layer homogeneous waveguide, the equivalent thickness for the tapered layer of Ta(2)O(5) and the sensitivity of the sensor to adlayer thickness were determined. Responses of the sensor to unspecific adsorption of bovine serum albumin (BSA) and to surface antibody-antigen interaction were investigated in situ and the corresponding surface coverages were obtained with the adlayer-thickness sensitivity. The interferometer sensor shows good long-term stability and its phase drift is lower than π over 10 h.

Objective: In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation., Methods: Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide., Results: The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope., Conclusion: The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures., (Copyright © 2011 by the American College of Rheumatology.)

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.

Context-awareness is a characteristic in the recognition between antigens and antibodies, highlighting the reconfiguration of epitope residues when an antigen interacts with a different antibody. A coarse binary classification of antigen regions into epitopes, or nonepitopes without specifying antibodies may not accurately reflect this biological reality. Therefore, we study an antibody-specified epitope prediction problem in line with this principle. This problem is new and challenging as we pinpoint a subset of the antigenic residues from an antigen when it binds to a specific antibody. We introduce two kinds of associations of the contextual awareness: 1) residues-residues pairing preference, and 2) the dependence between sets of contact residue pairs. Preference plays a bridging role to link interacting paratope and epitope residues while dependence is used to extend the association from one-dimension to two-dimension. The paratope/epitope residues' relative composition, cooperativity ratios, and Markov properties are also utilized to enhance our method. A nonredundant data set containing 80 antibody-antigen complexes is compiled and used in the evaluation. The results show that our method yields a good performance on antibody-specified epitope prediction. On the traditional antibody-ignored epitope prediction problem, a simplified version of our method can produce a competitive, sometimes much better, performance in comparison with three structure-based predictors.

An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed., (Copyright © 2011 John Wiley & Sons, Ltd.)

In this paper, we report an immunoassay in which probe proteins are immobilized on the surface of liquid crystal (LC) droplets rather than on solid surfaces. The advantage of this immunoassay is that the binding of antibodies to the probe proteins can be transduced by the LC droplets directly without the need for additional steps. For example, when we incubate the LC droplets decorated with immunoglobulin G (IgG) in a solution containing anti-IgG (AIgG), these droplets change their orientations from radial to bipolar configuration. In contrast, when we incubate the IgG-LC droplets in a solution containing anti-human serum albumin (AHSA), no changes are observed. The change of orientational configuration indicates the formation of the antigen-antibody immunocomplex on the surface of the LC droplets. Using LC droplet immunoassays, we successfully detect antibody concentrations as low as 0.01 μg/mL for AIgG and 0.02 μg/mL for AHSA. Because the immunoassay using LC droplets is label-free and gives a unique optical response, it has the potential to be further developed as a portable and low-cost immunoassay., (© 2011 American Chemical Society)

The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization., (© 2011 International Union of Crystallography. All rights reserved.)

We have developed a new fluorescent immune ensemble probe comprised of a conjugated lower toxic water-soluble CdTe:Zn(2+) quantum dots (QDs) and Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2)- antibody complex (Ru-Ab) for the dual-color determination of human enterovirus 71 (EV71) in homogeneous solution. EV71 monoantibody was easily covalently conjugated with Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2) to form a stable complex Ru-Ab, which acted both as an effective quencher of QDs fluorescence and the capture probe of virus antigen EV71. Herein, the target EV71 can break up the low fluorescent ionic ensemble by antigen-antibody combination to set free the fluorescent QDs and restore the fluorescence of QDs whereas the fluorescence intensity of Ru-Ab remains the same. Thus, the determination of EV71 by the complex Ru-Ab and QDs can be realized via the restoration of QDs fluorescence upon addition of EV71 and even can be directly evaluated by the ratio of green-colored QDs fluorescence intensity to Ru-Ab red-colored fluorescence intensity. The green-colored fluorescence of QDs was very sensitive to the change of EV71 concentration, and its fluorescence intensity increased with the increase of EV71 concentration between 1.8 ng/mL and 12 μg/mL. With this method, EV71 was detected at subnanogram per milliliter concentration in the presence of 160 μg/mL bovine serum albumin. More importantly, this strategy can be used as a universal method for any protein or virus by changing conjugated antibodies in disease early diagnosis providing a fast and promising clinical approach for virus determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 has been described. It could be applied in real sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

A microchip fluorescence-enhanced immunoassay method was developed for simultaneous detection of carcinoma antigen 125 (CA125) and carbohydrate antigen 15-3 (CA15-3). In this method, CA125 and CA15-3 react with excess amount of fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (Ab(*)) of CA125 and CA15-3 to form CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes. Microchip electrophoresis (MCE) separation of free Ab(125)(*), Ab(15-3)(*), and CA125-Ab(125)(*), CA15-3-Ab(15-3)(*) complexes were then performed. The separated species were sensitively detected by laser-induced fluorescence detection (LIF). CA125 and CA15-3 were quantified simultaneously by measuring the fluorescence intensity of CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes, respectively. Under the optimum conditions, the limits of detection were 0.23 U/mL for CA125 and 0.09 U/mL for CA15-3. The present MCE-LIF method was applied to the determination of CA125 and CA15-3 in serum from healthy subjects and cancer patients. The levels of CA125 and CA15-3 in these sera samples were found to be in the ranges of 15.6-36.1 U/mL and 13.8-28.4 U/mL for healthy subjects, and 192.5-368.3 U/mL and 63.3-198.4 U/mL for cancer patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen-monoclonal antibody (Ag-mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen-antibody complex in solution.

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb&#95;PrP&#95;01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb&#95;PrP&#95;01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 Å, β = 101.96° and diffracted to 2.1 Å resolution using synchrotron radiation. Human PrP (90-231)/Nb&#95;PrP&#95;01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 Å, β = 96.23° and diffracted to 1.5 Å resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.

Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

Computational protein design methods can complement experimental screening and selection techniques by predicting libraries of low-energy sequences compatible with a desired structure and function. Incorporating backbone flexibility in computational design allows conformational adjustments that should broaden the range of predicted low-energy sequences. Here, we evaluate computational predictions of sequence libraries from different protocols for modeling backbone flexibility using the complex between the therapeutic antibody Herceptin and its target human epidermal growth factor receptor 2 (HER2) as a model system. Within the program RosettaDesign, three methods are compared: The first two use ensembles of structures generated by Monte Carlo protocols for near-native conformational sampling: kinematic closure (KIC) and backrub, and the third method uses snapshots from molecular dynamics (MD) simulations. KIC or backrub methods were better able to identify the amino acid residues experimentally observed by phage display in the Herceptin-HER2 interface than MD snapshots, which generated much larger conformational and sequence diversity. KIC and backrub, as well as fixed backbone simulations, captured the key mutation Asp98Trp in Herceptin, which leads to a further threefold affinity improvement of the already subnanomolar parental Herceptin-HER2 interface. Modeling subtle backbone conformational changes may assist in the design of sequence libraries for improving the affinity of antibody-antigen interfaces and could be suitable for other protein complexes for which structural information is available., (Copyright © 2011 The Protein Society.)

The features of monovalent and bivalent binding of receptors (or antibodies) with a polyvalent ligand (or with an antigen) are considered. It is shown that the rigid connection of the binding sites of the receptor brings to high increase of binding affinity for the corresponding ligand, but only in case if its epitopes are fully complementary to both sites of the receptor binding. If not, then there is no advantage of the binding of bivalent receptor before univalent binding. If the binding sites of the receptor are connected by a flexible linker, then regardless of location of epitopes of the corresponding ligand there is the successful fastening of receptor and ligand. Exactly the connection by a flexible linker is used by Nature in most cases at constructing of polyvalent receptors.

We report the synthesis and in vitro evaluation of a multivalent homing device, a polymer which contains preordered pendant groups with dual specificity, a trisaccharide moiety, which is specific for the siglec CD22, and an antibody specific hapten, nitrophenol. The device efficiently attracts antihapten IgM to the surface of human lymphoma B cells as well as to CD22-conjugated magnetic beads by mediating the formation of a ternary complex on the surface of the target.

IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only FcγRI is capable of IgG binding with high affinity. FcγRI exists as a complex of a ligand binding α-chain and an FcR γ-chain. The receptors' α-chain can, furthermore, elicit several functions independent of the ITAM-bearing FcR γ-chain. Functional implications of high-affinity IgG binding and mechanisms underlying FcR γ-chain-independent signaling remain unclear to this day. In this paper, we provide an overview of past literature on FcγRI and address the implications of recently described interactions between cytosolic proteins and the FcγRI α-chain, as well as cytokine-enhanced FcγRI immune complex binding. Furthermore, an analysis of potential polymorphisms within the FCGR1A gene is provided.

A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1.9 Å resolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å(2)) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Å resolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with 'lock and key' binding. The binding arrangements in the TSHR260-K1-70 Fab complex are similar to previously observed for the TSHR260-M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260-K1-70 and TSHR260-M22 complexes show a root mean square deviation on all C(α) atoms of only 0.51 Å. These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.

The L1CAM antibody A10-A3 efficiently reduces tumor growth in a nude mouse model. Here, we describe the crystal structure of the Fab fragment of A10-A3 determined at 2.0 angstrom resolution. The A10-A3 antibody H3 loop reveals a characteristic arrangement of exposed aromatic residues that may play an important role in antigen binding. A structure model of the complex between L1CAM Ig1-4 and A10-A3 Fab indicates that the Fab binds to three small loops outside Ig1 and a residue between Ig1 and Ig2, consistent with an epitope mapping result. The data presented here should contribute to the design of high-affinity antibody for therapeutic purposes as well as to the understanding of neural cell remodeling and cancer progression mechanism mediated by L1CAM., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121-125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies., (Copyright © 2010 John Wiley & Sons, Ltd.)

Computational modeling techniques and computer simulations have become a routine in biological sciences and have gained great attention from researchers. Molecular dynamics simulation is a valuable tool towards an understanding of the complex structure of biological systems, especially in the study of the flexibility of the biological molecules such as peptides or proteins. Peptides play a very important role in human physiology and control many of the processes involved in the immune system response. Designing new and optimal peptide vaccines is one of the hottest challenges of the 21(st) century science and it brings together researchers from different fields. Molecular dynamics simulations have proven to be a helpful tool assisting laboratory work, saving financial sources and opening possibilities for exploring properties of the molecular systems that are hardly accessible by conventional experimental methods. Present review is dedicated to the recent contributions in applications of molecular dynamics simulations in peptide design for immunological purposes, such as B or T cell epitopes.

The tripartite protein exotoxin secreted by Bacillus anthracis, a major contributor to its virulence and anthrax pathogenesis, consists of binary complexes of the protective antigen (PA) heptamer (PA63h), produced by proteolytic cleavage of PA, together with either lethal factor or edema factor. The mouse monoclonal anti-PA antibody 1G3 was previously shown to be a potent antidote that shares F(C) domain dependency with the human monoclonal antibody MDX-1303 currently under clinical development. Here we demonstrate that 1G3 instigates severe perturbation of the PA63h structure and creates a PA supercomplex as visualized by electron microscopy. This phenotype, produced by the unconventional mode of antibody action, highlights the feasibility for optimization of vaccines based on analogous structural modification of PA63h as an additional strategy for future remedies against anthrax.

The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate.

An antibody Fab fragment, AbD1556, was selected against the extracellular domain of BMP receptor type IA, which blocks the binding of BMP-2 to BMPR-IA and thereby neutralizes BMP-2 activity. To study the mechanism by which BMPR-IA is recognized and bound by the Fab fragment, the complex of AbD1556 bound to BMPR-IA was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group P2(1), with unit-cell parameters a=89.32, b=129.25, c=100.24 A, beta=92.27 degrees.

Purpose: To analyze contribution of short aggregation-prone regions (APRs), which may self-associate via cross-beta motif and were earlier identified in therapeutic mAbs, towards antigen recognition via structural analyses of antibody-antigen complexes., Methods: A dataset of 29 publically available high-resolution crystal structures of Fab-antigen complexes was collected. Contribution of APRs towards the surface areas of the Fabs buried by the cognate antigens was computed. Propensities of amino acids to occur in APRs and to be involved in antigen binding were compared. Coincidence between APRs and individual CDR loops was examined., Results: All Fabs in the dataset contain at least one APR in CDR loops and adjacent framework beta-strands. The average contribution of APRs towards buried surface area of Fabs is 16.0 +/- 10.7%. Aggregation and antigen recognition may be coupled via aromatic residues (Tyr, Trp), which occur with high propensities in both APRs and antigen binding sites. APRs are infrequent in the heavy chain CDR 3 (H3) loops (7%), but are frequent in H2 loops (45%)., Conclusions: Co-incidence of APRs with antigen recognition sites can potentially lead to the loss of function upon aggregation. Rational structure-based design or selection strategies are suggested for biotherapeutics with improved druggability while maintaining potency.

Background: Accurate prediction of antigenic epitopes is important for immunologic research and medical applications, but it is still an open problem in bioinformatics. The case for discontinuous epitopes is even worse - currently there are only a few discontinuous epitope prediction servers available, though discontinuous peptides constitute the majority of all B-cell antigenic epitopes. The small number of structures for antigen-antibody complexes limits the development of reliable discontinuous epitope prediction methods and an unbiased benchmark to evaluate developed methods., Results: In this work, we present two novel server applications for discontinuous epitope prediction: EPSVR and EPMeta, where EPMeta is a meta server. EPSVR, EPMeta, and datasets are available at http://sysbio.unl.edu/services., Conclusion: The server application for discontinuous epitope prediction, EPSVR, uses a Support Vector Regression (SVR) method to integrate six scoring terms. Furthermore, we combined EPSVR with five existing epitope prediction servers to construct EPMeta. All methods were benchmarked by our curated independent test set, in which all antigens had no complex structures with the antibody, and their epitopes were identified by various biochemical experiments. The area under the receiver operating characteristic curve (AUC) of EPSVR was 0.597, higher than that of any other existing single server, and EPMeta had a better performance than any single server - with an AUC of 0.638, significantly higher than PEPITO and Disctope (p-value < 0.05).

The recombinant N-terminal domain of human ephrin type-A receptor 2 (rEphA2) has been crystallized in complex with the recombinantly produced Fab fragment of a fully human antibody (1C1; IgG1/kappa). These are the first reported crystals of an ephrin receptor bound to an antibody. The orthorhombic crystals belonged to space group C222(1) (the 00l reflections obey the l = 2n rule), with unit-cell parameters a = 78.93, b = 120.79, c = 286.20 A. The diffraction of the crystals extended to 2.0 A resolution. However, only data to 2.55 A resolution were considered to be useful owing to spot overlap caused by the long unit-cell parameter. The asymmetric unit is most likely to contain two 1C1 Fab-rEphA2 complexes. This corresponds to a crystal volume per protein weight (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49.5%. The three-dimensional structure of this complex will shed light on the molecular basis of 1C1 specificity. This will also contribute to a better understanding of the mechanism of action of this antibody, the current evaluation of which as an antibody-drug conjugate in cancer therapy makes it a particularly interesting case study.

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen." Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.

Background: Predicting B-cell epitopes is very important for designing vaccines and drugs to fight against the infectious agents. However, due to the high complexity of this problem, previous prediction methods that focus on linear and conformational epitope prediction are both unsatisfactory. In addition, antigen interacting with antibody is context dependent and the coarse binary classification of antigen residues into epitope and non-epitope without the corresponding antibody may not reveal the biological reality. Therefore, we take a novel way to identify epitopes by using associations between antibodies and antigens., Results: Given a pair of antibody-antigen sequences, the epitope residues can be identified by two types of associations: paratope-epitope interacting biclique and cooccurrent pattern of interacting residue pairs. As the association itself does not include the neighborhood information on the primary sequence, residues' cooperativity and relative composition are then used to enhance our method. Evaluation carried out on a benchmark data set shows that the proposed method produces very good performance in terms of accuracy. After compared with other two structure-based B-cell epitope prediction methods, results show that the proposed method is competitive to, sometimes even better than, the structure-based methods which have much smaller applicability scope., Conclusions: The proposed method leads to a new way of identifying B-cell epitopes. Besides, this antibody-specified epitope prediction can provide more precise and helpful information for wet-lab experiments.

We present the results of our comprehensive study of precipitation pattern formation by interacting immunogenic proteins in a gel medium. Formation of immunoprecipitation patterns was studied both theoretically and experimentally. Based on a system of reaction-diffusion equations, continuous deterministic description provides a quantitative model of reaction kinetics. Discrete stochastic microscopic description was used to supplement the results of reaction-diffusion model by mimicking product aggregation that contributes to a deeper understanding of the mechanism that governs the phenomenon. Our studies have shown that the mechanism of immunoprecipitation pattern formation is specific for protein precipitation and differs from such mechanisms for any inorganic or biological substances. By microscopic examination, we demonstrated that immunoprecipitation patterns can have a microstructure. We found that the microscopic structure of immunoprecipitation patterns results from multicomponent composition of antiserum., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The best understood mechanisms of omalizumab are that it neutralizes free IgE and down-regulates high-affinity IgE.Fc receptors (FcepsilonRI) on basophils and mast cells. It has been proposed that since complexes of IgE and omalizumab are accumulated to 5-10 times the basal levels of IgE, they may trap incoming allergens, contributing to omalizumab's effectiveness. In order to investigate the ability of IgE:omalizumab complexes in trapping allergens and inhibiting basophil activation in an in vitro reconstitution model, the ability of IgE:omalizumab complexes to tie up antigen and hence inhibit (a) antigen binding to IgE bound by FcepsilonRI, and (b) antigen-mediated activation of basophils, was examined. The free IgE was prepared by mixing different proportions of antigen-nonspecific IgE secreted by U266 cells and antigen-specific IgE, SE44 IgE, which recognizes a synthetic 15 a.a. peptide, R15K. The antigen was (R15K)(8)-ova, i.e. ovalbumin conjugated with an average of 8 copies of R15K per molecule. The solid-phase FcepsilonRI was a recombinant protein representing the extracellular portion of the alpha chain of the FcepsilonRI receptor complex. The model FcepsilonRI(+) basophilic cell line was RBL.SX-38, a rat basophilic leukemic line transfected with the genes for alpha, beta and gamma subunits of human FcepsilonRI. The results showed that the IgE:omalizumab complexes trapped increasing amounts of antigen with increasing (a) concentration of IgE, (b) proportion of antigen-specific IgE in total IgE, and (c) concentration of total immune complexes. Such trapping decreased the antigen-induced activation of FcepsilonRI(+) cells that had been pulsed with antigen-specific IgE, resulting in decreased mediator release. These results suggest that the rapidly accumulated IgE:omalizumab complexes in omalizumab-treated patients can capture allergens and consequently contribute to the pharmacological effects of omalizumab., (Copyright 2010 Elsevier B.V. All rights reserved.)

If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition. To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution. As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

High resolution structures of antibody-antigen complexes are useful for analyzing the binding interface and to make rational choices for antibody engineering. When a crystallographic structure of a complex is unavailable, the structure must be predicted using computational tools. In this work, we illustrate a novel approach, named SnugDock, to predict high-resolution antibody-antigen complex structures by simultaneously structurally optimizing the antibody-antigen rigid-body positions, the relative orientation of the antibody light and heavy chains, and the conformations of the six complementarity determining region loops. This approach is especially useful when the crystal structure of the antibody is not available, requiring allowances for inaccuracies in an antibody homology model which would otherwise frustrate rigid-backbone docking predictions. Local docking using SnugDock with the lowest-energy RosettaAntibody homology model produced more accurate predictions than standard rigid-body docking. SnugDock can be combined with ensemble docking to mimic conformer selection and induced fit resulting in increased sampling of diverse antibody conformations. The combined algorithm produced four medium (Critical Assessment of PRediction of Interactions-CAPRI rating) and seven acceptable lowest-interface-energy predictions in a test set of fifteen complexes. Structural analysis shows that diverse paratope conformations are sampled, but docked paratope backbones are not necessarily closer to the crystal structure conformations than the starting homology models. The accuracy of SnugDock predictions suggests a new genre of general docking algorithms with flexible binding interfaces targeted towards making homology models useful for further high-resolution predictions.

Many recombinant transcription factors have been invented, but we cannot select a substance used as an inducer. In this study, we have created a novel expression control system in which we can select a substance as an inducer toward which a monoclonal antibody (mAb) is prepared. The variable region fragments (Fvs) of the heavy and light chains (V(H) and V(L)) of the bisphenol A (BPA)-specific mAb BBA-2187 were each fused to the DNA-binding domain (DBD) of LexA and the transactivation domain (AD) of VP16. The association between the two recombinant proteins in the presence of BPA constituted a functional transcription factor. The recombinant proteins in which the DBD was fused to the N-terminal side of the Fv and in which the nuclear localization signal (NLS) was fused to the N-termini of the construct including the AD highly induced beta-galactosidase (lacZ) expression in recombinant yeast cells grown with BPA. When the Fvs of the polychlorinated biphenyl (PCB)-specific mAb 4444 were used, DBD-NLS-V(H) and NLS-AD-V(L) showed significantly increased lacZ activity in response to a PCB derivative. The Fv transcription factor may be useful in many fields such as gene therapeutics.

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide abeta(1-40). In epitope excision, the Fab and abeta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-abeta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, abeta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to abeta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-abeta(1-40) mAbs.

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 A resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca(2+). The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.