Block Antibody Binding Research Articles

The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

Simultaneous Measurement of IgM and IgG Antibodies to SARS-CoV-2 Spike, RBD, and Nucleocapsid Multiplexed in a Single Assay on the xMAP INTELLIFLEX DR-SE Flow Analyzer.

ABSTRACTThe multiplex capabilities of the new xMAP INTELLIFLEX DR-SE flow analyzer were explored by modifying a serological assay previously used to characterize the IgG antibody to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The goal was to examine the instrument’s performance and to simultaneously measure IgM and IgG antibody responses against multiple SARS-CoV-2 antigens in a single assay. Specific antibodies against the SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins were investigated in 310 symptomatic case patients using a fluorescent microsphere immunoassay and simultaneous detection of IgM and IgG. Neutralization potential was studied using the addition of soluble angiotensin-converting enzyme 2 (ACE2) to block antibody binding. A profile extending to 180 days from symptom onset (DFSO) was described for antibodies specific to each viral antigen. Generally, IgM levels peaked and declined rapidly ∼3–4 weeks following infection, whereas S- and RBD-specific IgG plateaued at 80 DFSO. ACE2 more effectively prevented IgM and IgG binding in convalescent cases > 30 DFSO, suggesting those antibodies had greater neutralization potential. This work highlighted the multiplex and multi-analyte potential of the xMAP INTELLIFLEX DR-SE, and provided further evidence for antigen-specific IgM and IgG trajectories in acute and convalescent cases.IMPORTANCE The xMAP INTELLIFLEX DR-SE enabled simultaneous and semi-quantitative detection of both IgM and IgG to three different SARS-CoV-2 antigens in a single assay. The assay format is advantageous for rapid and medium-throughput profiling using a small volume of specimen. The xMAP INTELLIFLEX DR-SE technology demonstrated the potential to include numerous SARS-CoV-2 antigens; future work could incorporate multiple spike protein variants in a single assay. This could be an important feature for assessing the serological response to emerging variants of SARS-CoV-2.

The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

Brucellosis is a global disease and the world’s most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.

Mouse Norovirus Uses Host Metabolites to Enhance Receptor Binding and Evade Immune Recognition

Noroviruses are the major cause of epidemic gastroenteritis in humans, causing ~20 million cases annually, resulting in more than 70,000 hospitalizations and 570-800 deaths in the United States alone. The T=3 icosahedral calicivirus capsid is composed of viral protein 1 (VP1) with three major domains: the N-terminus (N), shell (S), and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Mouse norovirus (MNV) is a widely used system for study of norovirus biology since we have a cell culture system, reverse genetic tools, and small animal model to eventually correlate structural information to whole animal pathology Mouse norovirus is a surprisingly dynamic virus that switches between receptor and antibody binding structures depending upon the in-vivo environment. In the circulation, the P domain floats above the shell by more than 15Å and the P domain loops (A’B’/E’F’) at the very tip are splayed apart in an ‘open’ conformation that antibodies learn to recognize. Upon ingestion, the low pH environment with high metal and bile salt concentrations in the alimentary canal each independently trigger the P domains to rotate 90° and contract by 15 Å onto the capsid surface. This hides any epitopes at base of the P domain. During this reversible collapse, the two P domains within the dimer rotate about each other and the A’B’/E’F’ loops adopt the ‘closed’ conformation. This opens the receptor binding site while burying the epitopes at the tip of the P domain. Therefore, rather than only depending on escape mutations to block antibody binding, MNV aggressively uses host conditions to remodel itself to enhance receptor binding while blocking antibody recognition. This review will describe the structural processes and biological consequences of the virus responding to activating host cues in the gut while these same triggers bury the epitopes presented in the circulation. This is an aggressive and unique mode of immune escape that has been subsequently shown in other viruses such as COVID-19. Therefore, a deeper understanding of the dynamic processes of virus capsids will improve vaccine design by understanding how to present the epitope conformations at the site of infection rather than what is presented to the immune system.

Polyethylene Glycol Immunogenicity: Theoretical, Clinical, and Practical Aspects of Anti-Polyethylene Glycol Antibodies.

Polyethylene glycol (PEG) is a flexible, hydrophilic simple polymer that is physically attached to peptides, proteins, nucleic acids, liposomes, and nanoparticles to reduce renal clearance, block antibody and protein binding sites, and enhance the half-life and efficacy of therapeutic molecules. Some naïve individuals have pre-existing antibodies that can bind to PEG, and some PEG-modified compounds induce additional antibodies against PEG, which can adversely impact drug efficacy and safety. Here we provide a framework to better understand PEG immunogenicity and how antibodies against PEG affect pegylated drug and nanoparticles. Analysis of published studies reveals rules for predicting accelerated blood clearance of pegylated medicine and therapeutic liposomes. Experimental studies of anti-PEG antibody binding to different forms, sizes, and immobilization states of PEG are also provided. The widespread use of SARS-CoV-2 RNA vaccines that incorporate PEG in lipid nanoparticles make understanding possible effects of anti-PEG antibodies on pegylated medicines even more critical.

Multiple Signals in the Gut Contract the Mouse Norovirus Capsid To Block Antibody Binding While Enhancing Receptor Affinity

Human norovirus is the leading cause of gastroenteritis worldwide, with no approved vaccine or antiviral treatment to mitigate infection. These plus-strand RNA viruses have T = 3 icosahedral protein capsids with 90 pronounced protruding (P) domain dimers, to which antibodies and cellular receptors bind. We previously demonstrated that bile binding to the capsid of mouse norovirus (MNV) causes several major conformational changes; the entire P domain rotates by ∼90° and contracts onto the shell, the P domain dimers rotate about each other, and the structural equilibrium of the epitopes at the top of the P domain shifts toward the closed conformation, which favors receptor binding while blocking antibody binding. Here, we demonstrate that MNV undergoes reversible conformational changes at pH 5.0 that are nearly identical to those observed when bile binds. Notably, at low pH or when metals bind, a cluster of acidic resides in the G'-H' loop interact and distort the G'-H' loop, and this may drive C'-D' loop movement toward the closed conformation. Enzyme-linked immunosorbent assays with infectious virus particles at low pH or in the presence of metals demonstrated that all tested antibodies do not bind to this contracted form, akin to what was observed with the MNV-bile complex. Therefore, low pH, cationic metals, and bile salts are physiological triggers in the gut for P domain contraction and structural rearrangement, which synergistically prime the virus for receptor binding while blocking antibody binding. IMPORTANCE The protruding domains on the calicivirus capsids are recognized by cell receptors and antibodies. We demonstrated that MNV P domains are highly mobile, and bile causes contraction onto the shell surface while allosterically blocking antibody binding. We present the near-atomic cryo-electron microscopy structures of infectious MNV at pH 5.0 and pH 7.5. Surprisingly, low pH is sufficient to cause the same conformational changes as when bile binds. A cluster of acidic residues on the G'-H' loop were most likely involved in the pH effects. These residues also bound divalent cations and had the same conformation as observed here at pH 5. Binding assays demonstrated that low pH and metals block antibody binding, and thus the G'-H' loop might be driving the conformational changes. Therefore, low pH, cationic metals, and bile salts in the gut synergistically prime the virus for receptor binding while blocking antibody binding.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Adsorptive mutation and N-linked glycosylation modulate influenza virus antigenicity and fitness

ABSTRACT Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is “fit” and able to overcome vaccine pressure.

Linear epitope binding antibodies against GII.3 Norovirus exhibit no histo-blood group antigens (HBGAs) blocking effects.

Noroviruses are leading cause of acute gastroenteritis worldwide. In our previous study, we established an in vitro histo-blood group antigens (HBGAs) binding blockade assay against GII.3 Norovirus virus like particles (VLPs) with trypsin digestion. In this study, we characterized the blocking antibody binding site and epitope type (linear or conformational) by using hyperimmune sera produced against different antigens. VP1 from Jingzhou402 (GII.3, JZ402) strain was expressed by using pGEX-6p-1 expression vector and the insoluble proteins were purified for immunization in rabbit. Previously characterized chimeric VP1-assembled VLPs (GII.4-VP1/GII.3-P2) were used to immunize guinea pig. Peptides reactive with hyperimmune serum against VLPs derived from the VP1 of JZ402 strain were conjugated with BSA and used to immunize rabbits. Hyperimmune sera against above antigens and JZ402 and JZ403 strain-derived VLPs were used to compare their HBGAs blocking effects. Rabbit anti-GST-VP1 and BSA-peptide conjugated hyperimmune sera demonstrated no blocking effects against the binding of GII.3 and GII.4 NoV VLPs to salivary HBGAs. Guinea pig anti-GII.4-VP1/GII.3-P2 hyperimmune serum blocked the binding of trypsin cleaved GII.3 VLPs to salivary HBGAs with no or very weak blocking effects against the binding of GII.4 VLPs to salivary HBGAs. Our data indicated that HBGAs blocking antibodies primarily bound the P2 domain of GII.3 NoV VP1 and their binding epitopes were most probably conformation-dependent.

Effects of pre-analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels.

The use of pre-analytical heat treatment (PHT) with the Nijmegen-Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30minutes and heating to 58°C for 90minutes. Data examining the effects of PHT on anti-FVIII IgG4 , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited. To assess the effect of PHT on the levels of detectable anti-FVIII IgG4 . Nijmegen-Bethesda assay-positive specimens were incubated at 56, 58 or 60°C for 90minutes, and anti-FVIII IgG4 was measured by fluorescence immunoassay (FLI) at 30-minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested. Levels of anti-FVIII IgG4 showed little change following incubations at 56°C (mean 101% of original value at 30minutes and 100% at 60minutes) but decreased upon exposure to 58°C (mean 85% at 30minutes and 66% at 60minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII. The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

Highlights of This Issue

In some colorectal cancer (CRC) patients who develop resistance to cetuximab, tumors have acquired somatic mutations that map to the cetuximab epitope, blocking antibody binding. Bagchi, Haidar and colleagues show that necitumumab, another FDA approved EGFR antibody, binds to and inhibits cetuximab resistant EGFR, despite sharing an almost identical epitope to cetuximab. Unlike cetuximab, necitumumab has a large paratope cavity that accommodates CRC-linked substitutions in EGFR, such as the commonly observed S468R (S492R including the signal peptide). A simple computational approach predicts which EGFR variants bind necitumumab. This work suggests that necitumumab may be effective in some cetuximab-resistant CRC cases.Esophageal adenocarcinoma (EAC) is inherently heterogeneous and often resistant to therapy. Hippo co-activator YAP1 is amplified in EAC and highly expressed. YAP1 confers cancer stem cell (CSC) properties, aggressive phenotype, and therapy resistance. Targeting YAP1 would be a novel therapeutic strategy but YAP1 inhibitors are currently lacking. In this issue, Song and colleagues report identification of CA3 as a potent YAP1 inhibitor. CA3 inhibited YAP1/Tead transcriptional activity and the growth of YAP1-high and radiation resistant EAC cells endowed with CSCs properties. When combined with fluorouracil, the therapeutic effects of CA3 were amplified both in vitro and in vivo.TRK kinase activation by chromosomal rearrangement is an oncogenic driver in a small percentage of tumors across a wide range of histologies. Effective targeted therapy against TRK has had significant positive impact in these patients' lives. Building upon preclinical and clinical studies demonstrating entrectinib efficacy in TRK fusion-positive solid tumors, this report provides evidence of entrectinib efficacy in TRK fusion-positive AML models. This study has broad implications in hematologic malignancies, with NTRK fusions identified in ALL, CEL and MM. As molecular profiling increases in hematologic malignancies, it is increasingly important to investigate options for patients with rare but actionable alterations.The clinical use of progesterone receptor (PR)-targeted therapies is limited, in part, by the lack of biomarkers that predict response to PR-agonists or PR-antagonists. Understanding the molecular mechanisms of action will provide an advance in developing novel combination therapies for cancer patients. Huang, Hu and colleagues have identified that onapristone, a pure PR antagonist, inhibited nuclear translocation of ligand-dependent or EGF-induced phospho-PR (S294), while trametinib only inhibited nuclear translocation of EGF induced phospho-PR (S294). Moreover, the combination of onapristone and trametinib resulted in superior anti-tumor effects in uterine cancer models, highlighting that targeting MAPK-dependent PR activation with onapristone and trametinib may be worthy of further development.

Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms.

Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region-one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from-is therefore ideal for vaccine development.

Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry.

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.

Overcoming Resistance to Anti-EGFR Therapy in Colorectal Cancer.

Our understanding of the genetic and nongenetic molecular alterations associated with anti-epidermal growth factor receptor (EGFR) therapy resistance in colorectal cancer (CRC) has markedly expanded in recent years. Mutations in RAS genes (KRAS/NRAS exons 2, 3, or 4) predict a lack of clinical benefit when anti-EGFR monoclonal antibodies (mAbs) are added to chemotherapy. Genetic events in additional nodes of the mitogen-activated protein kinase (MAPK)-phosphoinositide 3-kinase (PI3K) pathways that bypass EGFR signaling, such as BRAF or PIK3CA mutations or KRAS, ERBB2, or MET amplifications, also may confer resistance to cetuximab or panitumumab. Polymorphisms that block antibody binding as a result of EGFR extracellular domain mutations have been reported. Nongenetic mechanisms, including compensatory activation of receptor tyrosine kinases HER3 and MET, together with high expression of the ligands amphiregulin, transforming growth factor alpha heregulin, and hepatocyte growth factor in the tumor microenvironment also are thought to be involved in resistance. In one-third of the samples, more than one genetic event can be found, and nongenetic events most likely coexist with gene alterations. Furthermore, activation of a gene expression signature of epithelial-mesenchymal transition has been associated with reduced cellular dependence on EGFR signaling. Collectively, this body of work provides convincing evidence that the molecular heterogeneity of CRC plays an important role in the context of resistance to anti-EGFR therapy. Herein, we discuss how this knowledge has been translated into drug development strategies to overcome primary and acquired anti-EGFR resistance, with rational combinations of targeted agents in genomically selected populations, second-generation EGFR inhibitors, and other agents expected to boost the immune response at the tumor site.

Failure to detect anti-idiotypic antibodies in the autoimmune response to IA-2 in Type 1 diabetes

The concept that immune responses to self antigens are regulated by anti-idiotypic networks has attracted renewed interest following reports of circulating factors within IgG fractions of serum that impair detection of autoantibodies with autoantigen. Thus, preclearance of sera with bead-immobilised monoclonal autoantibodies to the Type 1 diabetes autoantigen GAD65, or prebinding of serum antibodies to protein A Sepharose prior to addition of antigen, increases immunoreactivity detected in serum samples consistent with the trapping on the beads of anti-idiotypic antibodies that block antibody binding to the autoantigen. The aim of this study was to investigate the presence of anti-idiotypic antibodies to another major target of autoantibodies in Type 1 diabetes, IA-2. As previously observed for GAD65, preadsorption of serum samples with immobilised monoclonal IA-2 autoantibody, or prebinding to protein A Sepharose, resulted in substantial increases in subsequent immunoprecipitation of radiolabeled IA-2 in a proportion of samples. However, control experiments indicated that the increases seen on pre-incubation with immobilized autoantibodies were caused by displacement of the antibody by serum IgG, whereas impaired detection of immunoreactivity in liquid-phase radiobinding assays was the result of formation of insoluble complexes that bind poorly to protein A. The results emphasise the importance of direct demonstration of specific binding of antibodies to the idiotype in the study of idiotypic networks in autoimmunity. Variability between patients in formation of insoluble immune complexes has implications for the design and standardization of autoantibody assays for diabetes prediction.

Identification of Immunoreactive Tumour Antigens Using Free and Exosome-Associated Humoral Responses

Altered tumour antigens can initiate cellular and humoral immune responses; however, they often fail to eliminate tumours. In humans, the presence of cancer is generally associated with the suppression of T cell activation and effector responses, characterized as a Th1 to Th2 biased response. This Th2 response leads to the production of tumour-reactive antibodies. Further, neoplastic lesions and biological fluids of cancer patients contain an abundance of tumour-derived exosomes (TDE) expressing tumour antigens. Expression of tumour antigens on TDE may represent an antibody target and serve to block antibody binding to the tumour, implicating a role for these nanovesicles in tumour survival. In this study, ovarian tumour cell proteins were separated by two-dimensional electrophoresis (2-DE) and patient-derived antibodies were used to analyse immunoreactivity. Common immunoreactive proteins among ovarian cancer patients were identified by mass spectrometry and six proteins were selected based on recognitio...

Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody

We adapted the method of epitope mapping by site-directed masking, which was described for purified soluble antigens [Paus,D. and Winter,G. (2006) Proc. Natl Acad. Sci. USA, 103, 9172-9177.], to map the binding site of an inhibitory monoclonal antibody on the cell surface protein ecto-nucleotidase NTPDase3. Using homology modeling, we built a 3D structure of NTPDase3 and designed 21 single cysteine mutations distributed over the surface of the enzyme. The mutant proteins were expressed in cells, biotinylated with a cysteine-specific reagent, and then extracted with detergent and immobilized on streptavidin-coated plates. Tethering NTPDase3 via cysteine residues located in a surface patch near the active site cleft masked the epitope and blocked antibody binding, as evaluated by enzyme inhibition assay and by ELISA. We then constructed 18 single alanine substitution mutations within the defined patch and found that W403A, D414A, E415A and R419A decreased the inhibitory effect of the antibody, whereas the double mutation W403A/R419A abolished both antibody binding and enzyme inhibition, suggesting the critical role of these residues for interaction with the antibody. Lack of competition between the antibody and a non-hydrolyzable substrate analog AMPPCP, as well as location of the epitope adjacent to the active site, suggest a noncompetitive mechanism of inhibition by steric hindrance. The described technique should be useful for systematic epitope mapping in cell membrane proteins for which either a 3D structure is available, or a sufficiently accurate 3D model can be obtained by homology modeling.

The Effect of the CXCR4 Inhibitors, AMD3100 and AMD3465, in Multiple Myeloma Cell Homing and Its Interaction with the Bone Marrow Microenvironment.

Abstract 1826Poster Board I-852Patients with multiple myeloma (MM) typically present with the disease spread diffusely throughout the bone marrow (BM). This fact points to the role MM cell trafficking plays in disease progression. Like normal plasma cells, MM cell trafficking is directed by the cytokine, SDF-1 and its receptor, CXCR4. Using the small bicyclam molecule, AMD3100, to block the SDF-1/CXCR4 interaction, and in vivo flow cytometry to measure circulation time of MM cells in a xenograft model, we have previously shown that homing of injected AMD3100-treated MM cells to the bone marrow is perturbed. Soon after injection fewer AMD3100-treated than untreated MM cells are detected in mouse skull BM using in vivo fluorescence confocal microscopy. Furthermore, the combined treatment of established tumors with AMD3100 and bortezomib enhanced survival. Here we present data that the 2nd generation SDF-1/CXCR4 small monomacrocyclic inhibitor, AMD3465 behaves like its predecessor in blocking antibody binding to CXCR4 on MM cells, migration of MM cells toward SDF-1, and in vivo homing, but at concentrations 10-50 fold lower than AMD3100. Incubation with either 1uM AMD3465 or 50uM AMD3100 reduced antibody binding to CXCR4 on MM cells to isotype control levels. Likewise, treatment with AMD3465 reduced MM1S migration to 15% of that of untreated cells in a transwell migration assay. These in vitro effects translated in vivo into longer circulation times for AMD3465-treated MM cells than control cells. In vivo flow cytometry revealed that 40-50% of AMD3465-pre-treated cells remained in the circulation one hr after injection whereas untreated cells depleted to 20% of their original circulating cell count by that time. Future experiments will further describe the effect of AMD 3465 on MM cells in the BM microenvironment to answer whether AMD3465 can be used to mobilize MM cells from the BM environment or enhance survival when used in conjunction with therapeutic drugs. DisclosuresGhobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Update on the cloning of monoclonal anti‐desmoglein antibodies from human pemphigus patients: implications for targeted therapy

Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.

Antibody evasion by the N terminus of murid herpesvirus-4 glycoprotein B

Herpesviruses characteristically transmit infection from immune hosts. Although their success in escaping neutralization by pre-formed antibody is indisputable, the underlying molecular mechanisms remain largely unknown. Glycoprotein B (gB) is the most conserved component of the herpesvirus entry machinery and its N terminus (gB-NT) is a common neutralization target. We used murid herpesvirus-4 to determine how gB-NT contributes to the virus–antibody interaction. Deleting gB-NT had no obvious impact on virus replication, but paradoxically increased virion neutralization by immune sera. This reflected greater antibody access to neutralization epitopes on gH/gL, with which gB was associated. gB-NT itself was variably protected against antibody by O-linked glycans; on virions from epithelial cells it was protected almost completely. gB-NT therefore provides a protective and largely protected cover for a vulnerable part of gH/gL. The conservation of predicted glycosylation sites in other mammalian herpesvirus gB-NTs suggests that this evasion mechanism is widespread. Interestingly, the gB-NT glycans that blocked antibody binding could be targeted for neutralization instead by a lectin, suggesting a means of therapeutic counterattack.