Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

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Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

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Kathryn L Armour

Full Text Available We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.

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Langone, J J; Das, C; Mainwaring, R; Shearer, W T

1985-01-01

Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.

A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

International Nuclear Information System (INIS)

Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

2010-01-01

Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

Sudan ebolavirus long recovered survivors produce GP-specific Abs that are of the IgG1 subclass and preferentially bind FcγRI.

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Radinsky, Olga; Edri, Avishay; Brusilovsky, Michael; Fedida-Metula, Shlomit; Sobarzo, Ariel; Gershoni-Yahalom, Orly; Lutwama, Julius; Dye, John; Lobel, Leslie; Porgador, Angel

2017-07-20

Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors' sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.

Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.

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Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre

2011-02-15

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

Comparative studies on Fc receptors for IgG on resting and activated T lymphocytes

International Nuclear Information System (INIS)

Hueckel, C.; Jensen, H.L.; Rychly, J.; Sandor, M.; Erdei, A.; Gergely, J.

1986-01-01

Fc-receptors for IgG (FcγR) on resting (i.e. freshly prepared) and mitogen (Con A) or alloantigen-activated mouse spleen T cells were compared using binding of different markers such as 125 I-labelled immune complexes, 125 I-labelled anti FcγR monoclonal antibody, FITC-labelled aggr. IgG and sheep erythrocytes covered with specific antibody (EA rosetting). C3b receptors were detected by rosetting with sheep erythrocytes covered with antibody and complement (EAC rosetting). The electrophoretic mobility of the cells without or after binding of aggr. IgG was also tested. Differences between resting and activated T cells were found: (1) After activation of T cells by mitogen or alloantigen, a proportion of FcγR-positive cells increased two to four times. (2) FcγR number per FcγR-positive cell seemed to be higher on activated then on resting cells. (3) FcγR-positive resting cells did not shed their FcγR upon incubation at 4 0 C followed by incubation at 37 0 C, but FcγR-positive activated cells shed a remarkable proportion of their FcγR on the same conditions. (4) Binding of aggr. IgG caused a decrease of electrophoretic mobility of activated but not resting cells. (5) FcγR-positive resting cells were also C3b receptor-positive, whereas FcγR-positive activated cells had no detectable C3b receptors. (author)

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

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Karasuyama Hajime

2011-04-01

Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

Generation and Characterization of an IgG4 Monomeric Fc Platform.

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Lu Shan

Full Text Available The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.

Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297.

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Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

2014-06-01

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

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Hasenhindl, Christoph; Traxlmayr, Michael W; Wozniak-Knopp, Gordana; Jones, Phil C; Stadlmayr, Gerhard; Rüker, Florian; Obinger, Christian

2013-10-01

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms.

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More, Apurva S; Toprani, Vishal M; Okbazghi, Solomon Z; Kim, Jae H; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

2016-02-01

As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue). Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

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Sundgren, Nathan C; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D; Tanigaki, Keiji; Yuhanna, Ivan S; Chambliss, Ken L; Mineo, Chieko; Shaul, Philip W

2015-02-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. © 2014 American Heart Association, Inc.

IgG Receptor FcγRIIB Plays a Key Role in Obesity-Induced Hypertension

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Sundgren, Nathan C.; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D.; Tanigaki, Keiji; Yuhanna, Ivan S.; Chambliss, Ken L.; Mineo, Chieko; Shaul, Philip W.

2015-01-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB+/+ mice developed obesity-induced hypertension, FcγRIIB−/− mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet–fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. PMID:25368023

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets

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Armour, Kathryn L; Smith, Cheryl S; Turner, Craig P; Kirton, Christopher M; Wilkes, Anthony M; Hadley, Andrew G; Ghevaert, Cedric; Williamson, Lorna M; Clark, Michael R

2014-01-01

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions. PMID:24285214

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

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Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

2012-12-18

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

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Chung, Amy; Makuba, Jenniffer M; Ndlovu, Bongiwe; Licht, Anna; Robinson, Hannah; Ramlakhan, Yathisha; Ghebremichael, Musie; Reddy, Tarylee; Goulder, Philip; Walker, Bruce; Ndung'u, Thumbi; Alter, Galit

2018-04-03

Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naïve participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4 counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Schoch, Angela; Larraillet, Vincent

2017-01-01

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism...... half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct...

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

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Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

2012-01-01

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

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Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

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Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

2011-01-01

Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS. Copyright © 2010 Elsevier B.V. All rights reserved.

The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region

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Rayner, Lucy E.; Hui, Gar Kay; Gor, Jayesh; Heenan, Richard K.; Dalby, Paul A.; Perkins, Stephen J.

2014-01-01

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s20,w0 of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration Rg was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron Rg values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser222) with extended hinge structures. The IgG4(Pro222) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications. PMID:24876381

Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

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Pollastrini, Joey; Dillon, Thomas M; Bondarenko, Pavel; Chou, Robert Y-T

2011-07-01

Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes. Copyright © 2011 Elsevier Inc. All rights reserved.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

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Se Jin Im

Full Text Available Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc consisting of IgD and IgG4, and tested its function using erythropoietin (EPO conjugate, EPO-hyFc. Despite low amino acid homology (20.5% between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H, a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last. Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

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Im, Se Jin; Yang, Sang In; Yang, Se Hwan; Choi, Dong Hoon; Choi, So Young; Kim, Hea Sook; Jang, Do Soo; Jin, Kyeong Sik; Chung, Yo-Kyung; Kim, Seung-Hee; Paik, Sang Hoon; Park, Yoo Chang; Chung, Moon Koo; Kim, Yong Bum; Han, Kang-Hyun; Choi, Kwan Yong; Sung, Young Chul

2011-01-01

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

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Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

1997-01-15

Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

Incorporation of FcRn-mediated disposition model to describe the population pharmacokinetics of therapeutic monoclonal IgG antibody in clinical patients.

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Ng, Chee M

2016-03-01

The two-compartment linear model used to describe the population pharmacokinetics (PK) of many therapeutic monoclonal antibodies (TMAbs) offered little biological insight to antibody disposition in humans. The purpose of this study is to develop a semi-mechanistic FcRn-mediated IgG disposition model to describe the population PK of TMAbs in clinical patients. A standard two-compartment linear PK model from a previously published population PK model of pertuzumab was used to simulate intensive PK data of 100 subjects for model development. Two different semi-mechanistic FcRn-mediated IgG disposition models were developed and First Order Conditional Estimation (FOCE) with the interaction method in NONMEM was used to obtain the final model estimates. The performances of these models were then compared with the two-compartment linear PK model used to simulate the data for model development. A semi-mechanistic FcRn-mediated IgG disposition model consisting of a peripheral tissue compartment and FcRn-containing endosomes in the central compartment best describes the simulated pertuzumab population PK data. This developed semi-mechanistic population PK model had the same number of model parameters, produced very similar concentration-time profiles but provided additional biological insight to the FcRn-mediated IgG disposition in human subjects compared with the standard linear two-compartment linear PK model. This first reported semi-mechanistic model may serve as an important model framework for developing future population PK models of TMAbs in clinical patients. Copyright © 2015 John Wiley & Sons, Ltd.

Associations between an IgG3 polymorphism in the binding domain for FcRn, transplacental transfer of malaria-specific IgG3, and protection against Plasmodium falciparum malaria during infancy: A birth cohort study in Benin.

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Celia Dechavanne

2017-10-01

Full Text Available Transplacental transfer of maternal immunoglobulin G (IgG to the fetus helps to protect against malaria and other infections in infancy. Recent studies have emphasized the important role of malaria-specific IgG3 in malaria immunity, and its transfer may reduce the risk of malaria in infancy. Human IgGs are actively transferred across the placenta by binding the neonatal Fc receptor (FcRn expressed within the endosomes of the syncytiotrophoblastic membrane. Histidine at position 435 (H435 provides for optimal Fc-IgG binding. In contrast to other IgG subclasses, IgG3 is highly polymorphic and usually contains an arginine at position 435, which reduces its binding affinity to FcRn in vitro. The reduced binding to FcRn is associated with reduced transplacental transfer and reduced half-life of IgG3 in vivo. Some haplotypes of IgG3 have histidine at position 435. This study examines the hypotheses that the IgG3-H435 variant promotes increased transplacental transfer of malaria-specific antibodies and a prolonged IgG3 half-life in infants and that its presence correlates with protection against clinical malaria during infancy.In Benin, 497 mother-infant pairs were included in a longitudinal birth cohort. Both maternal and cord serum samples were assayed for levels of IgG1 and IgG3 specific for MSP119, MSP2 (both allelic families, 3D7 and FC27, MSP3, GLURP (both regions, R0 and R2, and AMA1 antigens of Plasmodium falciparum. Cord:maternal ratios were calculated. The maternal IgG3 gene was sequenced to identify the IgG3-H435 polymorphism. A multivariate logistic regression was used to examine the association between maternal IgG3-H435 polymorphism and transplacental transfer of IgG3, adjusting for hypergammaglobulinemia, maternal malaria, and infant malaria exposure. Twenty-four percent of Beninese women living in an area highly endemic for malaria had the IgG3-H435 allele (377 women homozygous for the IgG3-R435 allele, 117 women heterozygous for the IgG

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

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Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

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Bereli, Nilay; Ertürk, Gizem; Tümer, M Aşkin; Say, Ridvan; Denizli, Adil

2013-05-01

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

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Gerco den Hartog

Full Text Available Respiratory syncytial virus (RSV infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.To investigate whether IgG purified from bovine milk (bIgG can modulate immune responses against human RSV.ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

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den Hartog, Gerco; Jacobino, Shamir; Bont, Louis; Cox, Linda; Ulfman, Laurien H; Leusen, Jeanette H W; van Neerven, R J Joost

2014-01-01

Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV. ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG

International Nuclear Information System (INIS)

Carson, D.A.; Lawrance, S.; Catalano, M.A.; Vaughan, J.H.; Abraham, G.

1977-01-01

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

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Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation

A vaccine of L2 epitope repeats fused with a modified IgG1 Fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types.

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Chen, Xue; Liu, Hongyang; Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

2014-01-01

Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17-36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.

Usefulness of FC-TRIPLEX Chagas/Leish IgG1 as confirmatory assay for non-negative results in blood bank screening of Chagas disease.

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Campos, Fernanda Magalhães Freire; Repoles, Laura Cotta; de Araújo, Fernanda Fortes; Peruhype-Magalhães, Vanessa; Xavier, Marcelo Antônio Pascoal; Sabino, Ester Cerdeira; de Freitas Carneiro Proietti, Anna Bárbara; Andrade, Mariléia Chaves; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Gontijo, Célia Maria Ferreira

2018-04-01

A relevant issue in Chagas disease serological diagnosis regards the requirement of using several confirmatory methods to elucidate the status of non-negative results from blood bank screening. The development of a single reliable method may potentially contribute to distinguish true and false positive results. Our aim was to evaluate the performance of the multiplexed flow-cytometry anti-T. cruzi/Leishmania IgG1 serology/(FC-TRIPLEX Chagas/Leish IgG1) with three conventional confirmatory criteria (ELISA-EIA, Immunofluorescence assay-IIF and EIA/IIF consensus criterion) to define the final status of samples with actual/previous non-negative results during anti-T. cruzi ELISA-screening in blood banks. Apart from inconclusive results, the FC-TRIPLEX presented a weak agreement index with EIA, while a strong agreement was observed when either IIF or EIA/IIF consensus criteria were applied. Discriminant analysis and Spearman's correlation further corroborates the agreement scores. ROC curve analysis showed that FC-TRIPLEX performance indexes were higher when IIF and EIA/IIF consensus were used as a confirmatory criterion. Logistic regression analysis further demonstrated that the probability of FC-TRIPLEX to yield positive results was higher for inconclusive results from IIF and EIA/IIF consensus. Machine learning tools illustrated the high level of categorical agreement between FC-TRIPLEX versus IIF or EIA/IIF consensus. Together, these findings demonstrated the usefulness of FC-TRIPLEX as a tool to elucidate the status of non-negative results in blood bank screening of Chagas disease. Copyright © 2018. Published by Elsevier B.V.

NRG1-Fc improves metabolic health via dual hepatic and central action.

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Zhang, Peng; Kuang, Henry; He, Yanlin; Idiga, Sharon O; Li, Siming; Chen, Zhimin; Yang, Zhao; Cai, Xing; Zhang, Kezhong; Potthoff, Matthew J; Xu, Yong; Lin, Jiandie D

2018-03-08

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues

DEFF Research Database (Denmark)

Andersen, Jan T; Justesen, Sune; Fleckenstein, Burkhard

2008-01-01

knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251......) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity...... was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response...

A strategy for bacterial production of a soluble functional human neonatal Fc receptor

DEFF Research Database (Denmark)

Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril

2008-01-01

The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus...

Intracellular calcium mobilization in human lymphocytes in the presence of synthetic IgG Fc peptides

International Nuclear Information System (INIS)

Plummer, J.M.; Panahi, Y.P.; McClurg, M.R.; Hahn, G.S.; Naemura, J.R.

1986-01-01

Certain synthetic peptides derived from the Fc region of human IgG can suppress the mixed lymphocyte response. These peptides were tested for the ability to induce intracellular calcium mobilization in human lymphocytes using fura-2/calcium fluorescence. T cells were isolated by rosetting and were > 90% OKT3 positive. Lymphocytes were incubated with the acetoxymethyl ester of fura-2 (10 μM) for 60 minutes at 37 0 C. Fluorescence intensity changes at 505 nm were monitored at an excitation lambda of 340 nm. Fura-2 was not cytotoxic compared to quin-2 since fura-2 loaded mononuclear cells incorporated 3 H-thymidine when stimulated by PHA, succinyl Con A, PWM or LPS-STM whereas quin-2 loaded cells showed a dose dependent inhibition of proliferation. Those synthetic peptides (5 to 400 μg/ml) that suppressed the MLR induced a dose dependent increase in intracellular calcium in mononuclear cells, lymphocytes, non-T cells and T cells. The fura-2 calcium fluorescence time course response was similar for peptide, PHA and succinyl Con A. These results suggest that these immunoregulatory peptides suppress 3 H-thymidine incorporation at a point after intracellular calcium mobilization and that fura-2 has advantages over quin-2 in measuring intracellular calcium levels in lymphocytes

Correlation of Fc(gamma)RIIa (CD32) Polymorphism and IgG Antibody Subclasses in Hemolytic Disease of Newborn.

Science.gov (United States)

Wu, QiangJu; Zhang, Yan; Liu, MengLi; Wang, Bo; Liu, Sheng; He, Chen

2009-01-01

ABO-HDN is a common disease of newborn in China and currently there is no satisfactory method to predict it in the antepartum period. It has been reported that Fc(gamma)RIIa (CD32) genotype is associated with both infectious diseases induced by bacteria and parasitemia. There is a relationship between IgG subclass and RH-HDN. To study the pathogenesis of ABO-HDN and to find reliable method to diagnose ABO-HDN, we investigated the polymorphism of Fc(gamma)RIIa (CD32) and distribution of IgG subclass in infants with ABO-HDN and their mothers by polymerase chain reaction or ELISA assay. We observed that the frequency of HH131 genotype is lower in infants with ABO-HDN than in controls (p < 0.01), while the frequency of HR131 genotype is higher in ABO-HDN infants than that in controls (p < 0.01). The genotype HR131 and concentrations of IgG1 and IgG3 are significantly correlated with ABO-HDN. Our results demonstrated, for the first time, that there is a correlation between ABO-HDN and CD32, and different IgG subclass distribution. Our study may contribute to the development of an early diagnostic method for HDN. Copyright 2009 S. Karger AG, Basel.

Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

Directory of Open Access Journals (Sweden)

Janina Jamasbi, RPh

2016-04-01

Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs1

Science.gov (United States)

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-01-01

Antibody-mediated rejection of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor specific antibody binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the antibody. We investigated the mechanisms underlying monocyte recruitment by HLA class I antibody-activated endothelium. We used a panel of murine monoclonal antibodies of different subclasses to crosslink HLA I on human aortic, venous and microvascular endothelial cells, and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. Mouse IgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during antibody mediated rejection. We confirmed these observations using human HLA allele specific monoclonal antibodies and IgG purified from transplant patient sera. HLA I antibodies universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during antibody-mediated rejection. Importantly, the subclass of donor specific antibody may influence its pathogenesis. These results imply that hIgG1 and hIgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions. PMID:23690477

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

Science.gov (United States)

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

2013-07-05

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

Science.gov (United States)

Gillis, Caitlin M; Jönsson, Friederike; Mancardi, David A; Tu, Naxin; Beutier, Héloïse; Van Rooijen, Nico; Macdonald, Lynn E; Murphy, Andrew J; Bruhns, Pierre

2017-04-01

Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

Science.gov (United States)

van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G

2001-04-15

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Science.gov (United States)

Castilho, Alexandra; Gruber, Clemens; Thader, Andreas; Oostenbrink, Chris; Pechlaner, Maria; Steinkellner, Herta; Altmann, Friedrich

2015-01-01

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF3, and GnGnF6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies. PMID:26067753

A simple solid-phase radioimmunoassay for the measurement of IgG secreted in vitro by human lymphocytes

International Nuclear Information System (INIS)

Romagnani, S.; Prete, G.F. del; Giudizi, G.M.; Almerigogna, F.; Ricci, M.

1979-01-01

A radioimmunoassay is described for measuring IgG, based on the ability of immunoglobulins of this class to inhibit the binding of radioiodinated staphylococcal protein A to IgG linked to a solid phase. The solid phase is represented by ox erythrocytes coated with anti-ox erthrocyte rabbit IgG, a reagent used for detecting cells equipped with receptors for the Fc fragment of IgG. By this assay the IgG secreted in vitro by human peripheral blood lymphocytes stimulated with PWM and those present in samples of very diluted human sera were measured. It was found that the assay is a very rapid, simple and and reproducible procedure for the detection of IgG immunoglobulin at the nanogram level. (Auth.)

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

Science.gov (United States)

Arora, Jayant; Hu, Yue; Esfandiary, Reza; Sathish, Hasige A; Bishop, Steven M; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Weis, David D

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

International Nuclear Information System (INIS)

Mueller, H.; Fehr, J.

1986-01-01

The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

Advances in therapeutic Fc engineering - modulation of IgG associated effector functions and serum half-life

Directory of Open Access Journals (Sweden)

Abhishek Saxena

2016-12-01

Full Text Available Today monoclonal immunoglobulin gamma (IgG antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs are achieved through both its antigen binding fragment (Fab and crystallizable fragment (Fc. Fab can specifically recognize tumor associated antigen (TAA and thus modulate TAA-linked downstream signaling pathways that may lead to inhibition of tumor growth, induction of tumor apoptosis and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC and antibody dependent cell-mediated phagocytosis (ADCP. Moreover, Fc is the region interacting with the neonatal Fc receptor (FcRn in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anti-cancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering based mAbs under clinical trials.

Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes

DEFF Research Database (Denmark)

Jimenez-Solem, Espen; Rasmussen, Mette H; Christensen, Mikkel

2010-01-01

Dulaglutide (LY-2189265) is a novel, long-acting glucagon-like peptide 1 (GLP-1) analog being developed by Eli Lilly for the treatment of type 2 diabetes mellitus (T2DM). Dulaglutide consists of GLP-1(7-37) covalently linked to an Fc fragment of human IgG4, thereby protecting the GLP-1 moiety from...

The Emerging Importance of IgG Fab Glycosylation in Immunity

NARCIS (Netherlands)

van de Bovenkamp, Fleur S.; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-01-01

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

Science.gov (United States)

Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Joing-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie

2014-06-01

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Science.gov (United States)

Mittal, Rahul; Sukumaran, Sunil K; Selvaraj, Suresh K; Wooster, David G; Babu, M Madan; Schreiber, Alan D; Verbeek, J Sjef; Prasadarao, Nemani V

2010-11-18

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Endothelial cells the site of the regulation of IgG LEVEL by salvaging and transcytosis of immunoglobulin

International Nuclear Information System (INIS)

Antohe, Felicia; Radulescu, Luminita; Simionescu, Maya; Ghetie, Victor

2002-01-01

basolateral surface, and in the reverse direction. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. An ex vivo placental model has been used to analyze the materno-fetal transfer of a recombinant, humanized (IgG1) antibody in which His435 has been mutated to alanine (H435A). The results showed that: a) FcRn is expressed by human placental endothelial cells in a functionally active form; b) transcytosis of IgG in HPEC is a time dependent process that takes place preferentially from the basolateral to the apical compartment; c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive; d) the binding of IgG to FcRn is a prerequisite for the transport across the placenta. Together, these data suggest that the regulation of IgG level by endothelial cell may result from interplay between endocytosis, exocytosis and transcytosis of the molecule. One can safely assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which endothelial cells maintain the plasma IgG homeostasis. (authors)

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Science.gov (United States)

Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs.

Science.gov (United States)

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-06-15

Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

Fc-mediated immune precipitation. III. Visualization by electron microscopy

DEFF Research Database (Denmark)

Møller, NPH; Christiansen, Gunna

1983-01-01

with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2...... fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes...

The Pathogenicity of Anti-β2GP1-IgG Autoantibodies Depends on Fc Glycosylation

Directory of Open Access Journals (Sweden)

Christoph Fickentscher

2015-01-01

Full Text Available To analyze the glycosylation of anti-β2GP1, we investigated purified IgG from healthy children, patients with APS, and asymptomatic adult carriers of antiphospholipid antibodies. We observed that in the sera of healthy children and of patients with APS, IgG3 and IgG2 were predominant, respectively. The potentially protective anti-β2GP1-IgM was lower in the sera of healthy children. Although anti-β2GP1-associated C1q did not differ between children and patients with antiphospholipid syndrome, the associated C3c was significantly higher in the sera of healthy children. This indicates a more efficient clearance of anti-β2GP1 immune complexes in the healthy children. This clearance is not accompanied by inflammation or coagulatory events. It is likely that the most important pathogenic factor of the anti-β2GP1-IgG is related to the different glycosylation observed in healthy and diseased individuals. We detected a significantly higher sialylation of anti-β2GP1-IgG isolated from the sera of healthy children and asymptomatic adults when compared with that of patients with clinically apparent antiphospholipid syndrome. Low sialylated IgG reportedly ameliorates inflammation and inflammation promotes hyposialylation. Thus, both reactions create a vicious circle that precipitates the pathology of the antiphospholipid syndrome including thrombus-formation. We conclude that the increased sialylation of anti-β2GP1-IgG of sera of healthy individuals limits their pathogenicity.

The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.

Science.gov (United States)

Sockolosky, Jonathan T; Szoka, Francis C

2015-08-30

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Science.gov (United States)

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

DEFF Research Database (Denmark)

Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje

2013-01-01

alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a non-destructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on two human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge...

Neuron-derived IgG protects dopaminergic neurons from insult by 6-OHDA and activates microglia through the FcγR I and TLR4 pathways.

Science.gov (United States)

Zhang, Jie; Niu, Na; Wang, Mingyu; McNutt, Michael A; Zhang, Donghong; Zhang, Baogang; Lu, Shijun; Liu, Yuqing; Liu, Zhihui

2013-08-01

Oxidative and immune attacks from the environment or microglia have been implicated in the loss of dopaminergic neurons of Parkinson's disease. The role of IgG which is an important immunologic molecule in the process of Parkinson's disease has been unclear. Evidence suggests that IgG can be produced by neurons in addition to its traditionally recognized source B lymphocytes, but its function in neurons is poorly understood. In this study, extensive expression of neuron-derived IgG was demonstrated in dopaminergic neurons of human and rat mesencephalon. With an in vitro Parkinson's disease model, we found that neuron-derived IgG can improve the survival and reduce apoptosis of dopaminergic neurons induced by 6-hydroxydopamine toxicity, and also depress the release of NO from microglia triggered by 6-hydroxydopamine. Expression of TNF-α and IL-10 in microglia was elevated to protective levels by neuron-derived IgG at a physiologic level via the FcγR I and TLR4 pathways and microglial activation could be attenuated by IgG blocking. All these data suggested that neuron-derived IgG may exert a self-protective function by activating microglia properly, and IgG may be involved in maintaining immunity homeostasis in the central nervous system and serve as an active factor under pathological conditions such as Parkinson's disease. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

Science.gov (United States)

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

Science.gov (United States)

Kapur, Rick; Della Valle, Luciana; Verhagen, Onno J H M; Hipgrave Ederveen, Agnes; Ligthart, Peter; de Haas, Masja; Kumpel, Belinda; Wuhrer, Manfred; van der Schoot, C Ellen; Vidarsson, Gestur

2015-03-01

RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction. Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history. HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%). RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered. © 2014 AABB.

Monovalent IgG4 molecules

Science.gov (United States)

Wilkinson, Ian C.; Fowler, Susan B.; Machiesky, LeeAnn; Miller, Kenneth; Hayes, David B.; Adib, Morshed; Her, Cheng; Borrok, M. Jack; Tsui, Ping; Burrell, Matthew; Corkill, Dominic J.; Witt, Susanne; Lowe, David C.; Webster, Carl I.

2013-01-01

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments. PMID:23567207

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

2015-01-01

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

FcγRll: Characterisation of novel Fc receptor interactions and a new receptor form.

OpenAIRE

JESSICA CLAIRE ANANIA

2018-01-01

Leukocyte Fc receptors (FcR) bind to immunogloulins (Ig) to link the innate and humoral immune system to help balance the immune system and clear infections. We have characterised a novel FcR for IgG (FcγR) form, designated FcγRIIa3, which contains a 19 amino acid insert. This insert interacts with cytoskeletal structures allowing the receptor to be retained for longer periods of time at the cells surface upon activation, higher cell signalling which causes greater cellular activation. Theref...

The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

Directory of Open Access Journals (Sweden)

Blaise Ndjamen

2014-03-01

Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

Science.gov (United States)

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

2016-01-01

The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Neuron-derived IgG protects neurons from complement-dependent cytotoxicity.

Science.gov (United States)

Zhang, Jie; Niu, Na; Li, Bingjie; McNutt, Michael A

2013-12-01

Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.

Fc gamma receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-gamma 1 and PLC-gamma 2 in natural killer cells

OpenAIRE

1992-01-01

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody- dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5- trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibit...

(Some) Cellular Mechanisms Influencing the Transcription of Human Endogenous Retrovirus, HERV-Fc1

DEFF Research Database (Denmark)

Laska, Magdalena Janina; Nissen, Kari Konstantin; Nexø, Bjørn Andersen

2013-01-01

DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well...... investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine...... and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza...

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

Science.gov (United States)

Kim, Geon A; Lee, Eun Mi; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Hwang, Jong Ik; Alam, Zahid; Ahn, Curie; Lee, Byeong Chun

2017-08-01

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG 1 -Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

Science.gov (United States)

Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

Science.gov (United States)

García-Montojo, Marta; Alcina, Antonio; Fedetz, María; Alloza, Iraide; Astobiza, Ianire; Leyva, Laura; Fernández, Oscar; Izquierdo, Guillermo; Antigüedad, Alfredo; Arroyo, Rafael; Álvarez-Lafuente, Roberto; Vandenbroeck, Koen; Matesanz, Fuencisla; Urcelay, Elena

2014-01-01

Background Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. Methods A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data. Results Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11–1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14–1.53)]. Conclusion Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts. PMID:24594754

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Science.gov (United States)

Hayes, Jerrard M; Frostell, Asa; Karlsson, Robert; Müller, Steffen; Martín, Silvia Míllan; Pauers, Martin; Reuss, Franziska; Cosgrave, Eoin F; Anneren, Cecilia; Davey, Gavin P; Rudd, Pauline M

2017-10-01

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIa Arg131/His131 (CD32a), RIIb (CD32b) and RIIIa Phe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIa Phe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIa Phe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a

Structural basis for pH-insensitive inhibition of immunoglobulin G recycling by an anti-neonatal Fc receptor antibody

Energy Technology Data Exchange (ETDEWEB)

Kenniston, Jon A.; Taylor, Brandy M.; Conley, Gregory P.; Cosic, Janja; Kopacz, Kris J.; Lindberg, Allison P.; Comeau, Stephen R.; Atkins, Kateri; Bullen, Jameson; TenHoor, Christopher; Adelman, Burt A.; Sexton, Daniel J.; Edwards, Thomas E.; Nixon, Andrew E. (Beryllium); (Dyax)

2017-09-06

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn–DX-2507 Fab complex, revealing a nearly complete overlap of the IgG–Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.

Human IgG1 antibodies suppress angiogenesis in a target-independent manner

NARCIS (Netherlands)

Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

2016-01-01

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression

Directory of Open Access Journals (Sweden)

Geon A. Kim

2017-01-01

Full Text Available Soluble human tumor necrosis factor (shTNFRI-Fc and human heme oxygenase 1 (hHO-1 are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

Science.gov (United States)

Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression.

Science.gov (United States)

Kim, Geon A; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Oh, Hyun Ju; Hwang, Joing-Ik; Ahn, Curie; Saadeldin, Islam M; Lee, Byeong Chun

2017-01-01

Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets ( P hHO-1 piglets ( P hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection

Science.gov (United States)

IgG is a major immunoglobulin subclass in mucosal secretions of human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about whether and how IgG enters the lumen of the genital tract and the exact role of local IgG may play ...

Bispecific engineered antibody domains (nanoantibodies that interact noncompetitively with an HIV-1 neutralizing epitope and FcRn.

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Rui Gong

Full Text Available Libraries based on an isolated human immunoglobulin G1 (IgG1 constant domain 2 (CH2 have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd (m01s with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn (Gong et al, JBC, 2009 and 2011. We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3 onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs. Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics.

Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

Science.gov (United States)

Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

2015-06-01

The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Conformational destabilization of Immunoglobulin G increases the low pH-binding affinity with the Neonatal Fc Receptor

DEFF Research Database (Denmark)

Walters, Benjamin T; Jensen, Pernille Foged; Larraillet, Vincent

2016-01-01

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the ......H-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.......Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain...... the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and Fc...

IgG4 subclass antibodies impair antitumor immunity in melanoma

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Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

2013-01-01

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

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Yijun Shen

Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using plasmagel and ficoll-hypaque.

Science.gov (United States)

Alexander, E L; Titus, J A; Segal, D M

1978-01-01

When mononuclear leukocytes are isolated directly from whole human blood using Ficoll-Hypaque or Plasmagel, cytophilic immunoglobulin is detected on cell surfaces. Upon incubation at 37 degrees C, this cell-associated immunoglobulin is shed slowly into the medium. However, when cells are prewashed in phosphate-buffered saline prior to isolation, they appear to be free of cytophilic immunoglobulin. Compared to prewashed cells, populations retaining cytophilic immunoglobulin on their surfaces demonstrate a decreased binding of soluble immune complexes and radiolabelled trimeric rabbit IgG. The data suggest that Ficoll-Hypaque and Plasmagel cause serum IgG to bind with abnormally high affinity to human mononuclear leukocytes, probably via Fc receptors. This artifact of preparation can lead to erroneous estimates of the numbers of cells bearing Fc receptors or intrinsic membrane immunoglobulin within a given population of cells and to an inaccurate assessment of the average number of Fc receptors per cell.

Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor

International Nuclear Information System (INIS)

Benach, J.L.; Fleit, H.B.; Habicht, G.S.; Coleman, J.L.; Bosler, E.M.; Lane, B.P.

1984-01-01

The phagocytic capacity of murine and human mononuclear and polymorphonuclear phagocytes (including peripheral blood monocytes and neutrophils), rabbit and murine peritoneal exudate cells, and the murine macrophage cell line P388D1 against the Lyme disease spirochete was studied. All of these cells were capable of phagocytosing the spirochete; phagocytosis was measured by the uptake of radiolabeled spirochetes, the appearance of immunofluorescent bodies in phagocytic cells, and electron microscopy. Both opsonized and nonopsonized organisms were phagocytosed. The uptake of opsonized organisms by neutrophils was blocked by a monoclonal antibody specific for the Fc receptor and by immune complexes; these findings suggested that most phagocytosis is mediated by the Fc receptor. Similarly, the uptake of opsonized organisms by human monocytes was inhibited by human monomeric IgG1 and by immune complexes. These results illustrate the role of immune phagocytosis of spirochetes in host defense against Lyme disease

Induction of functional Fc receptors in P388 leukemia cells. Requirement for multiple differentiation signals.

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Cohen, D A; Stotelmyer, N L; Kaplan, A M

1985-04-01

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.

Non-immune binding of human IgG to M-related proteins confers resistance to phagocytosis of group A streptococci in blood.

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Harry S Courtney

Full Text Available The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp are group A streptococcal (GAS receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.

Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

NARCIS (Netherlands)

Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules(1), Fab-arm exchange with therapeutic antibodies has not been

Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

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AM Marassá

2009-01-01

Full Text Available With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA. A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027 in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

NEW ROLES FOR FC RECEPTORS IN NEURODEGENERATION-THE IMPACT ON IMMUNOTHERAPY FOR ALZHEIMER’S DISEASE

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James P. Fuller

2014-08-01

Full Text Available There are an estimated 18 million Alzheimer’s disease (AD sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterised by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterised by deposits of amyloid beta (Aβ, neurofibrillary tangles, and neuroinflammation. Active immunisation or passive immunisation against Aβ leads to the clearance of deposits in transgenic mice expressing human Aβ. This clearance is associated with reversal of associated cognitive deficits, but these results have failed to translate to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aβ antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (Fcγ are a family of immunoglobulin like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking Fc receptors by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that Fc receptor expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. We propose that increased expression and ligation of Fc receptors in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of Fc receptors in the healthy and diseased brain.

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

Science.gov (United States)

Howie, Heather L; Delaney, Meghan; Wang, Xiaohong; Er, Lay See; Vidarsson, Gestur; Stegmann, Tamara C; Kapp, Linda; Lebedev, Jenna N; Wu, Yanyun; AuBuchon, James P; Zimring, James C

2016-12-01

Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported. © 2016 AABB.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Science.gov (United States)

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Activation of human natural killer cells by the soluble form of cellular prion protein

International Nuclear Information System (INIS)

Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

2015-01-01

Cellular prion protein (PrP C ) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP C in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP C protein on human natural killer (NK) cells. Recombinant soluble PrP C protein was generated by fusion of human PrP C with the Fc portion of human IgG 1 (PrP C -Fc). PrP C -Fc binds to the surface of human NK cells, particularly to CD56 dim NK cells. PrP C -Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP C -Fc facilitated the IL-15-induced proliferation of NK cells. PrP C -Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP C -Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP C (PrP C -Fc) was generated by fusion of human PrP C with IgG1 Fc portion. • PrP C -Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP C -Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

Science.gov (United States)

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

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Simone I Richardson

2018-04-01

Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

Science.gov (United States)

Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

2017-05-23

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

Science.gov (United States)

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Science.gov (United States)

Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Directory of Open Access Journals (Sweden)

Krishnamurthy Konduru

Full Text Available Ebola virus (EBOV, a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Science.gov (United States)

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.

Science.gov (United States)

Ikeda, Yuka; Kawai, Katsuhisa; Ikawa, Akira; Kawamoto, Kyoko; Egami, Youhei; Araki, Nobukazu

2017-08-01

Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup. © 2017. Published by The Company of Biologists Ltd.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

Science.gov (United States)

Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination

Directory of Open Access Journals (Sweden)

Sven Kratochvil

2017-12-01

Full Text Available Antibody subclasses exhibit extensive polymorphisms (allotypes that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1 of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC and antibody-dependent cellular phagocytosis (ADCP function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey.

Directory of Open Access Journals (Sweden)

Tsutomu Oshima

Full Text Available Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos. Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

International Nuclear Information System (INIS)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z_B_p_a–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z_B_p_a–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z_B_p_a–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL"-"1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL"-"1). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z_B_p_a–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z_B_p_a–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD of 2 ng mL"-"1 CEA

Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

Energy Technology Data Exchange (ETDEWEB)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao, E-mail: tangjb@wfmc.edu.cn

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z{sub Bpa}–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z{sub Bpa}–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z{sub Bpa}–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL{sup -1}, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL{sup -1}). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z{sub Bpa}–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z{sub Bpa}–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD

Fc-fusion Proteins in Therapy: An Updated View.

Science.gov (United States)

Jafari, Reza; Zolbanin, Naime M; Rafatpanah, Houshang; Majidi, Jafar; Kazemi, Tohid

2017-01-01

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

NARCIS (Netherlands)

Kapur, R.; Della Valle, L.; Verhagen, O.J.; Hipgrave Ederveen, A.; Ligthart, P.; de Haas, M.; Kumpel, B.; Wuhrer, M.; van der Schoot, C.E.; Vidarsson, G.

2015-01-01

Background RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D

NARCIS (Netherlands)

Kapur, Rick; Della Valle, Luciana; Verhagen, Onno J. H. M.; Hipgrave Ederveen, Agnes; Ligthart, Peter; de Haas, Masja; Kumpel, Belinda; Wuhrer, Manfred; van der Schoot, C. Ellen; Vidarsson, Gestur

2015-01-01

RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with

A novel TNFα antagonizing peptide-Fc fusion protein designed based on CDRs of TNFα neutralizing monoclonal antibody

International Nuclear Information System (INIS)

Qin Weisong; Feng Jiannan; Zhang Wei; Li Yan; Shen, Beifen

2004-01-01

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFα neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFα was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFα coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFα and also inhibit the TNFα-induced cytotoxicity on L929 cells. The TNFα antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFα antagonists

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

Science.gov (United States)

Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

2018-04-16

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

Science.gov (United States)

Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

2015-12-01

IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

A three-layer immunoradiometric assay for determination of IgG subclass antibodies in Human Sera (''IgG subclass RAST'')

International Nuclear Information System (INIS)

Djurup, R.; Soendergaard, I.; Weeke, B.; University of Copenhagen, Denmark); Magnusson, C.G.M.

1984-01-01

We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses, and the third layer is 125 I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgGI, anti-IgG3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-IgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Non-specific binding obtained with pooled normal human serum was below 0.33%. Inter-assay coefficient of variation was between 18 and 27%. The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy. (author)

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function.

Science.gov (United States)

Patel, Kashyap R; Roberts, Jacob T; Subedi, Ganesh P; Barb, Adam W

2018-03-09

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates ( N -glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N -glycans (23%). These proportions indicated restricted N -glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N -glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N -glycoforms similar to NK cell-derived CD16a but yielded N -glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N -glycan composition affected antibody binding: CD16a with oligomannose N -glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N -glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N -glycan composition and antibody-binding affinity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Macrophage colony stimulating factor (M-CSF) induces Fc receptor expression on macrophages

International Nuclear Information System (INIS)

Magee, D.M.; Wing, E.J.; Waheed, A.; Shadduck, R.K.

1986-01-01

M-CSF is a glycoprotein that stimulates bone marrow progenitor cells to proliferate and differentiate into macrophages (M theta). In addition, M-CSF can modulate the function of mature M theta. In this study, the authors determined the effect of M-CSF on expression of receptors for IgG (Fc receptors). Murine resident peritoneal M theta monolayers were incubated with either M-CSF, recombinant gamma interferon (IFN), or left untreated for 48 hrs. Expression of Fc receptors was assessed by microscopy using an antibody coated sheet erythrocytes (EA) rosette assay. The results indicated that M-CSF treated M theta had significantly higher numbers of bound EA (7.1 erythrocytes/M theta), than IFN M theta (4.4), or untreated M theta (2.5) (p 51 Cr labelled EA assay, CSF M theta (16,411 cpm), IFN M theta (10,887), untreated M theta (6897) (p < 0.001). Additionally, the maximal response was noted between 10 and 500 units M-CSF. Purified anti-M-CSF IgG, when included in the cultures, ablated the enhancement of EA binding, whereas normal rabbit IgG did not. These findings indicate that M-CSF is a potent inducer of Fc receptor expression on M theta and supports other data concerning the role of M-CSF as a biological response modifier

Human anti-rhesus D IgG1 antibody produced in transgenic plants

DEFF Research Database (Denmark)

Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed

2002-01-01

antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent......Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...... properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs....

Fc Gamma Receptor 3B (FCGR3Bc.233C>A-rs5030738) Polymorphism Modifies the Protective Effect of Malaria Specific Antibodies in Ghanaian Children

DEFF Research Database (Denmark)

Adu, Bright; Jepsen, Micha Phill Grønholm; Gerds, Thomas A

2014-01-01

Immunoglobulin G (IgG) cross-linking with Fc gamma receptor IIIB (FcγRIIIB) triggers neutrophil degranulation, releasing reactive oxygen species with high levels associated with protection against malaria. The FCGR3B-c.233C>A polymorphism thought to influence the interaction between IgG and Fcγ...

Age-dependent association between IgG2 and IgG3 subclasses to Pf332-C231 antigen and protection from malaria, and induction of protective antibodies by sub-patent malaria infections, in Daraweesh

DEFF Research Database (Denmark)

Giha, Hayder A; Nasr, Amre; Iriemenam, Nnaemeka C

2010-01-01

The certainty of the protective role of acquired immunity in malaria is the major drive for malaria vaccine development. In this study, we measured the levels of total IgG and IgG subclasses to four candidate malaria vaccine antigens; MSP2-3D7, MSP2-FC27, AMA-1 and Pf332-C231, in plasma obtained ...

Estimation of polyclonal IgG4 hybrids in normal human serum.

Science.gov (United States)

Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

2014-07-01

The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. © 2014 John Wiley & Sons Ltd.

Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias.

Science.gov (United States)

Mancuso, Maria Elisa; Mannucci, Pier Mannuccio

2014-01-01

Prophylaxis with regular infusions of factor VIII (FVIII)- or factor IX (FIX)- containing products is the mainstay of modern hemophilia care. However, this therapeutic regimen is inconvenient, requiring repeated intravenous injections from childhood. Approaches meant to prolong the half-life of FVIII and FIX in plasma have been developed in order to improve the feasibility and acceptability of replacement therapy, extending protection from bleeding, reducing infusion frequency and hence the need for venous access devices in young children. Several strategies have been implemented to enhance the pharmacokinetics of clotting factors, including conjugation with polyethylene glycol and the production by genetic engineering of fusion proteins containing the coagulation factors linked to a long-lived plasma protein such as albumin or the Fc fragment of immunoglobulin (Ig)G. The latter technology is one of the most promising, since the prolongation of FVIII and FIX half-life is obtained by exploiting the physiological binding of the Fc domain to the neonatal Fc receptor. Fc fusion monomers have been obtained with both recombinant FVIII (rFVIIIFc) and FIX (rFIXFc), and data from preclinical and clinical studies showed improved pharmacokinetics for both factors, which are produced in human embryonic kidney (HEK) 293 cells, thus ensuring full human post-translational modifications. In Phase I/IIa studies, rFVIIIFc and rFIXFc showed 1.5-1.7 fold and 3.0-4.0 fold longer elimination half-life, respectively. Similar data have been obtained in the Phase III clinical studies with rFVIIIFc and rFIX-Fc published recently. Both drugs were satisfactorily safe, particularly with respect to immunogenicity, and no serious adverse event was observed.

Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

NARCIS (Netherlands)

Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

2009-01-01

Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions.

Science.gov (United States)

Kinder, Michelle; Greenplate, Allison R; Strohl, William R; Jordan, Robert E; Brezski, Randall J

2015-01-01

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Science.gov (United States)

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

International Nuclear Information System (INIS)

Simms, H.H.; D'Amico, R.; Monfils, P.; Burchard, K.W.

1991-01-01

We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions

Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

Science.gov (United States)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

NARCIS (Netherlands)

Rispens, Theo; Davies, Anna M.; Ooijevaar-de Heer, Pleuni; Absalah, Samira; Bende, Onno; Sutton, Brian J.; Vidarsson, Gestur; Aalberse, Rob C.

2014-01-01

Background: Fab arm exchange requires weak interactions between CH3 domains, such as in human IgG4. Results: CH3-CH3 interactions differ >1,000,000-fold between human subclasses and allotypes due to variations Lys/Asn-392, Val/Met-397, and Lys/Arg-409. Conclusion: For IgG2 and IgG3, but not IgG1,

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers

NARCIS (Netherlands)

Lauc, G.; Huffman, J.E.; Pucic, M.; Zgaga, L.; Adamczyk, B.; Muzinic, A.; Novokmet, M.; Polasek, O.; Gornik, O.; Kristic, J.; Keser, T.; Vitart, V.; Scheijen, B.; Uh, H.W.; Molokhia, M.; Patrick, A.L.; McKeigue, P.; Kolcic, I.; Lukic, I.K.; Swann, O.; Leeuwen, F.N. van; Ruhaak, L.R.; Houwing-Duistermaat, J.J.; Slagboom, P.E.; Beekman, M.; Craen, A.J. de; Deelder, A.M.; Zeng, Q.; Wang, W.; Hastie, N.D.; Gyllensten, U.; Wilson, J.F.; Wuhrer, M.; Wright, A.F.; Rudd, P.M.; Hayward, C.; Aulchenko, Y.; Campbell, H.; Rudan, I.

2013-01-01

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative

TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Antitumor Activity and Minimal Erythrocyte Binding.

Science.gov (United States)

Petrova, Penka S; Viller, Natasja Nielsen; Wong, Mark; Pang, Xinli; Lin, Gloria H Y; Dodge, Karen; Chai, Vien; Chen, Hui; Lee, Vivian; House, Violetta; Vigo, Noel T; Jin, Debbie; Mutukura, Tapfuma; Charbonneau, Marilyse; Truong, Tran; Viau, Stephane; Johnson, Lisa D; Linderoth, Emma; Sievers, Eric L; Maleki Vareki, Saman; Figueredo, Rene; Pampillo, Macarena; Koropatnick, James; Trudel, Suzanne; Mbong, Nathan; Jin, Liqing; Wang, Jean C Y; Uger, Robert A

2017-02-15

Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47-SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication. Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails. Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo , TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies. Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068-79. ©2016 AACR . ©2016 American Association for Cancer Research.

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice.

Science.gov (United States)

Nogueira, Raquel Tayar; Sahi, Vincent; Huang, Jing; Tsuji, Moriya

2017-08-01

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Science.gov (United States)

Kim, Mi Young; Copland, Alastair; Nayak, Kaustuv; Chandele, Anmol; Ahmed, Muhammad S; Zhang, Qibo; Diogo, Gil R; Paul, Matthew J; Hofmann, Sven; Yang, Moon-Sik; Jang, Yong-Suk; Ma, Julian K-C; Reljic, Rajko

2017-12-09

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4 + and CD8 + T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life

DEFF Research Database (Denmark)

Bukrinski, Jens T.; Sønderby, Pernille; Antunes, Filipa

2017-01-01

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of less than 3 minutes limits its clinical...... use. A strategy to extend the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days owing to its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial...... with the available structural information on the FcRn and GLP-1 receptor (GLP-1R) obtained from X-ray crystallography, we can explain the observed in-vitro and in-vivo behaviour. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease...

Comparison of the chemical behaviour of humanized ACMS VS. Human IGG radiolabeled with 99mTc

International Nuclear Information System (INIS)

Rivero Santamaria, Alejandro; Zayas Crespo, Francisco; Mesa Duennas, Niurka; Castillo Vitloch, Adolfo J.

2003-01-01

The purpose of this work is to compare the chemical behaviour of humanized AcMs vs. human IgG radiolabeled with 99 mTc. to this end, 3 immunoglobulins were analyzed, the IgG (human), the humanized monoclonal antibody R3 (Acm-R3h) and the humanized monoclonal antibody T1. The results obtained reveal slight differences as regards the behaviour of theses immunoglobulins before the labelling with 99T c, which shows differences in the chemical behaviour of these proteins. Although in theory the modifications that are made to the AcMs in order to humanize them must not affect their chemical behaviour, the obtained data indicate that the conditions for their radiolabelling should not be extrapolated from other proteins; on the contrary, particular procedures should be elaborated for each AcM-h

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhenbao [Central South University, School of Pharmaceutical Sciences (China); Zhou, Bo, E-mail: zhoubo1771@163.com [The Affiliated Zhongda Hospital of Southeast University, Department of Gerontology (China); Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang, E-mail: lengxigyky@163.com [Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College (China)

2013-09-15

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 {mu}m in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL{sup -1} to 10 ng mL{sup -1}. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

Science.gov (United States)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

International Nuclear Information System (INIS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-01-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL −1 to 10 ng mL −1 . This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples

Avaliação das subclasses IgG1 e IgG3 na doença hemolítica perinatal Assessment of IgG1 and IgG3 subclasses in perinatal hemolytic disease

Directory of Open Access Journals (Sweden)

Maria A. Araújo

2003-01-01

Full Text Available A doença hemolítica perinatal (DHPN ainda é um problema clínico. Nenhum teste isolado prediz, com segurança, a gravidade do quadro hemolítico. O objetivo do presente estudo foi determinar as subclasses de anticorpos IgG1 e IgG3 por citometria de fluxo no soro de 42 gestantes isoimunizadas e correlacionar os dados obtidos com a gravidade da DHPN. A distribuição dos fetos ou neonatos segundo a gravidade do quadro hemolítico evidenciou 13 casos com doença leve, 16 casos com doença moderada e 13 com doença grave. As subclasses foram detectadas em 33/42 (79% amostras. A subclasse IgG1, isoladamente, foi evidenciada em 14/33 (42,4% casos. Na relação entre gravidade da doença e subclasses de IgG, observou-se que IgG1 isolada foi encontrada em todos os grupos, e os valores da mediana de intensidade de fluorescência (MIF foram significativamente mais altos nas formas mais graves da DHPN (pThe hemolytic disease of the newborn (HDN continues to be a clinical problem in spite of prophylaxis. To date, none of the available tests, developed to predict the severity of HDN, has provided complete reliability. The objective of the present study was to determine the IgG1 and IgG3 subclasses in 42 isoimmunized pregnant women, and to correlate them with clinical severity of hemolytic disease. The IgG subclasses were determined employing flow cytometry. According to the clinical severity of HDN, fetuses and newborn babies were classified as 13 mild, 16 moderate and 13 severe cases. The IgG subclasses were detected in 33 of the 42 pregnant women. Of these, IgG1 was predominant in 72.7% of the cases; either isolated (42.4% or in association with IgG3 (30.3%. IgG1 was present in all the three clinical severity categories, however, its values were significantly higher in cases with greater clinical severity of HDN (p<0.01. On the other hand, the distribution of IgG3 values within each group was not statistically significant (p=0.11. IgG3 seems to be more

The apparent monovalency of human IgG4 is due to bispecificity

NARCIS (Netherlands)

Aalberse, R. C.; Schuurman, J.; van Ree, R.

1999-01-01

A hypothesis is put forward to explain the apparent monovalency of human IgG4. It is based upon the known instability of the IgG4 hinge. IgG4 is secreted as a regular bivalent antibody, but after secretion interacts with another IgG4 molecule. This interaction results in the exchange of half

Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment.

Directory of Open Access Journals (Sweden)

Iain W Chalmers

Full Text Available The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29 containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study. While vaccination with SmLy6A (SmCD59a and SmLy6D (Sm29 induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored.Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K. Our examination extends the number of members to eleven (including three novel proteins and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE responses against two surface-bound representatives (SmLy6A and SmLy6B within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively, these values are both higher than IgG1 prevalence (2.7% for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively when compared to rising IgG

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131).

Science.gov (United States)

Cymer, Florian; Thomann, Marco; Wegele, Harald; Avenal, Cecile; Schlothauer, Tilman; Gygax, Daniel; Beck, Hermann

2017-11-01

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI

International Nuclear Information System (INIS)

Johnson, D.C.; Ligas, M.W.; Frame, M.C.; Cross, A.M.; Stow, N.D.

1988-01-01

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70. Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, the authors have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI. Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG

A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF.

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Ji, Jianjian; Xu, Jingjing; Li, Fanlin; Li, Xiaojing; Gong, Wei; Song, Yuxian; Dou, Huan; Hou, Yayi

2016-05-01

Myeloid dendritic cells (DCs) can produce B-cell-activating factor (BAFF) that modulates survival and differentiation of B cells and plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Toll-like receptor 4 (TLR4) signaling has important functions in the process of BAFF production. Our previous study showed that a benzenediamine derivate FC-99 possesses anti-inflammation activity and directly interacts with interleukin-1 receptor-associated kinase 4 (IRAK4), which was a pivotal molecule in TLR4 signaling. In this study, we demonstrated that FC-99 attenuated lupus nephritis in the MRL/lpr mice. FC-99 also decreased the levels of total immunoglobulin G (IgG), total IgG2a and IgM in sera, as well as the activation of B cells in the spleens of MRL/lpr mice. Moreover, FC-99 inhibited abnormal activation of myeloid DCs in spleens and reduced the levels of BAFF in sera, spleens, and kidneys of MRL/lpr mice. Furthermore, upon TLR4 stimulation with lipopolysaccharide in vitro, FC-99 inhibited IRAK4 phosphorylation, as well as the activation and BAFF production in murine bone marrow-derived DCs. These data indicate that FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting DC-secreted BAFF, suggesting that FC-99 may be a potential therapeutic candidate for the treatment of SLE. © The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Activation of human natural killer cells by the soluble form of cellular prion protein

Energy Technology Data Exchange (ETDEWEB)

Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

2015-08-21

Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis

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Yannic C. Bartsch

2018-06-01

Full Text Available Pro- and anti-inflammatory effector functions of IgG antibodies (Abs depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0 IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors, administered in high doses (2 g/kg to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.

Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fc gamma RIIIa (CD16)

NARCIS (Netherlands)

Kumpel, BM; De Haas, M; Koene, HR; Van de Winkel, JGJ; Goodrick, MJ

Human red cells (RBC) coated with IgG anti-D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcgamma R). Polymorphisms of Fcgamma RIIa and Fcgamma RIIIa affect IgG binding in vitro , and may alter the efficiency of clearance of immune complexes in vivo. In

Inhibition of th17 cells and promotion of tregs in fc gamma chain-deficient mice contributes to the attenuated atherosclerotic lesions

Science.gov (United States)

The presence of anti-oxLDL IgG is well documented in clinical and animal studies. However, the role for Fc Rs to the progression of atherosclerosis has not been studied in detail. In the present study, we investigated the role for activating Fc R in the progression of atherosclerosis using apoE-Fc -...

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Science.gov (United States)

Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

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Omar Rafael Alemán

2016-01-01

Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Chicken IgY Fc expressed by Eimeria mitis enhances the immunogenicity of E. mitis.

Science.gov (United States)

Qin, Mei; Tang, Xinming; Yin, Guangwen; Liu, Xianyong; Suo, Jingxia; Tao, Geru; Ei-Ashram, Saeed; Li, Yuan; Suo, Xun

2016-03-21

Eimeria species are obligate intracellular apicomplexan parasites, causing great economic losses in the poultry industry. Currently wild-and attenuated- type anticoccidial vaccines are used to control coccidiosis. However, their use in fast growing broilers is limited by vaccination side effects caused by medium and/or low immunogenic Eimeria spp. There is, therefore, a need for a vaccine with high immunogenicity for broilers. The avian yolk sac IgY Fc is the avian counterpart of the mammalian IgG Fc, which enhances immunogenicity of Fc-fusion proteins. Here, we developed a stable transgenic Eimeria mitis expressing IgY Fc (Emi.chFc) and investigated whether the avian IgY Fc fragment enhances the immunogenicity of E. mitis. Two-week-old broilers were immunized with either Emi.chFc or wild type Eimeria and challenged with wild type E. mitis to analyze the protective properties of transgenic Emi.chFc. Chickens immunized with Emi.chFc had significantly lower oocyst output, in comparison with PBS, mock control (transgenic E. mitis expressing HA1 from H9N2 avian influenza virus) and wildtype E. mitis immunized groups after challenge, indicating that IgY Fc enhanced the immunogenicity of E. mitis. Our findings suggest that IgY Fc-expressing Eimeria may be a better coccidiosis vaccine, and transgenic Eimeria expressing Fc-fused exogenous antigens may be used as a novel vaccine-delivery vehicle against a wide variety of pathogens.

Quantitative cumulative biodistribution of antibodies in mice

Science.gov (United States)

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level. PMID:24572100

Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

International Nuclear Information System (INIS)

Martins, Estefania M.N.; Andrade, Antero S.R.; Reis, Bernardo S.; Goes, Alfredo M.

2007-01-01

Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

Dynamics evaluation of total IgG, IgG1 and IgG2a in the serum of mice immunized with radioattenuated paracoccidioides brasiliensis yeast cells

Energy Technology Data Exchange (ETDEWEB)

Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)]. E-mail: estefaniabio@yahoo.com.br; antero@cdtn.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia]. E-mail: brsgarbi@mono.icb.ufmg.br; goes@mono.icb.ufmg.br

2007-07-01

Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans. Up to the moment no vaccine has still been reported. The potential of gamma radiation for pathogens attenuation and vaccine development was explored in this work. In our laboratory we developed radioattenuated yeast cells of P. brasiliensis and the aim of the present work was to evaluate the antibody production dynamics in mice immunized with this cells. Were analyzed the IgG antibodies titers as well as the type of response by analyzing the IgG1 and IgG2a antibody pattern in the course of infection. The mice were divided in two groups that were immunized one time and two times respectively. The mice infected with the virulent P. brasiliensis showed a high level of antibody production while the infection with the radioattenuated yeast did not significantly change the antibody level. The level of IgG raised in both immunized groups after the challenge. In the group immunized one time was not observed a significant difference between the levels of both subclasses when compared with the control. After the challenge of the group immunized two times the IgG2a levels increased significantly when analyzed 90 days post challenge. We concluded that a pattern related to the disease control was apparent in the group submitted to two immunizations. The mice had not developed a totally polarized pattern of TH1/TH2 response but a trend to a TH1 response was evident. (author)

Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

Science.gov (United States)

Hui, James Z; Tsourkas, Andrew

2014-09-17

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

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Eleonora Dehlink

Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

Application of hanging drop technique to optimize human IgG formulations.

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Li, Guohua; Kasha, Purna C; Late, Sameer; Banga, Ajay K

2010-01-01

The purpose of this work is to assess the hanging drop technique in screening excipients to develop optimal formulations for human immunoglobulin G (IgG). A microdrop of human IgG and test solution hanging from a cover slide and undergoing vapour diffusion was monitored by a stereomicroscope. Aqueous solutions of IgG in the presence of different pH, salt concentrations and excipients were prepared and characterized. Low concentration of either sodium/potassium phosphate or McIlvaine buffer favoured the solubility of IgG. Addition of sucrose favoured the stability of this antibody while addition of NaCl caused more aggregation. Antimicrobial preservatives were also screened and a complex effect at different buffer conditions was observed. Dynamic light scattering, differential scanning calorimetry and size exclusion chromatography studies were performed to further validate the results. In conclusion, hanging drop is a very easy and effective approach to screen protein formulations in the early stage of formulation development.

LatY136F knock-in mouse model for human IgG4-related disease.

Science.gov (United States)

Yamada, Kazunori; Zuka, Masahiko; Ito, Kiyoaki; Mizuguchi, Keishi; Kakuchi, Yasushi; Onoe, Tamehito; Suzuki, Yasunori; Yamagishi, Masakazu; Izui, Shozo; Malissen, Marie; Malissen, Bernard; Kawano, Mitsuhiro

2018-01-01

The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (LatY136F) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of LatY136F knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD). LatY136F knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed. Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment. LatY136F knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the LatY136F knock-in mouse

Chicken IgY Fc linked to Bordetella avium ompA and Taishan Pinus massoniana pollen polysaccharide adjuvant enhances macrophage function and specific immune responses

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Zhu Ruiliang

2016-11-01

Full Text Available Fc-fusion technologies, in which immunoglobulin Fc is genetically fused to an antigenic protein, have been developed to confer antibody-like properties to proteins and peptides. Mammalian IgG Fc fusion exhibits improved antigen-induced immune responses by providing aggregates with high avidity for the IgG Fc receptor and salvaging the antigenic portion from endosomal degradation. However, whether the linked chicken IgY Fc fragment shares similar characteristics to mammalian IgG Fc remains unclear. In this study, we linked the chicken IgY Fc gene to the outer membrane protein A (ompA of Borderella avium through overlapping PCR. The fusion gene was cloned into the pPIC9 plasmid to construct the recombinant Pichia pastoris transformant expressing the ompA–Fc fusion protein. The effects of the linked Fc on macrophage vitality, activity, efficiency of antigen processing, and immune responses induced by the fused ompA were investigated. Furthermore, the effect of Taishan Pinus massoniana pollen polysaccharide (TPPPS, an immunomodulator, on chicken macrophage activation was evaluated. TPPPS was also used as an adjuvant to investigate its immunomodulatory effect on immunoresponses induced by the fused ompA–Fc in chickens. The pinocytosis, phagocytosis, secretion of nitric oxide and TNF-α, and MHC-II molecular expression of the macrophages treated with the fused ompA–Fc were significantly higher than those of the macrophages treated with ompA alone. The addition of TPPPS to the fused ompA–Fc further enhanced macrophage functions. The fused ompA–Fc elicited higher antigen-specific immune responses and protective efficacy compared with ompA alone. Moreover, the fused ompA–Fc conferred higher serum antibody titers, serum IL-2 and IL-4 concentrations, CD4+ and CD8+ T-lymphocyte counts, lymphocyte transformation rate, and protection rate compared with ompA alone. Notably, the prepared TPPPS adjuvant ompA–Fc vaccines induced high immune

A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

Science.gov (United States)

Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

2015-03-20

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

OpenAIRE

Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

2016-01-01

Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

OpenAIRE

Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

2017-01-01

Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

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Afridi Sarwat

2012-09-01

Full Text Available Abstract Background HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. Methods In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson’s correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. Results After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i haemoglobin C with IgG levels; ii the FcγRIIa H/R131 with IgG2 and IgG3 levels; iii TNF-863 with IgG3 levels; iv TNF-857 with IgG levels; and, v TNF1304 with IgG3, IgG4, and IgG levels. Conclusion Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual’s humoral response against malaria.

Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

Science.gov (United States)

Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

2015-05-01

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Genetic association of multiple sclerosis with the marker rs391745 near the endogenous retroviral locus HERV-Fc1: analysis of disease subtypes

DEFF Research Database (Denmark)

Hansen, Bettina; Oturai, Annette Bang; Harbo, Hanne F

2011-01-01

We have previously described the occurrence of multiple sclerosis (MS) to be associated with human endogenous retroviruses, specifically the X-linked viral locus HERV-Fc1. The aim of this study was to investigate a possible association of the HERV-Fc1 locus with subtypes of MS. MS patients......-Fc1 locus (p = 0.003), while primary progressive disease was not. The ability to see genetic differences between subtypes of MS near this gene speaks for the involvement of the virus HERV-Fc1 locus in modifying the disease course of MS....

A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

Science.gov (United States)

Li, Dan; He, Wenhui; Liu, Ximing; Zheng, Sanduo; Qi, Yonghe; Li, Huiyu; Mao, Fengfeng; Liu, Juan; Sun, Yinyan; Pan, Lijing; Du, Kaixin; Ye, Keqiong; Li, Wenhui; Sui, Jianhua

2017-09-26

Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

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Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

Science.gov (United States)

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

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Hirani Nikhil

2010-10-01

Full Text Available Abstract Background A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF. The R131H (rs1801274 polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression. Methods In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification. Results No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC and lower diffusing capacity for carbon monoxide (DLCO. Similarly, increased frequency of the H131 allele was observed in patients with severe disease (DLCO 10% drop in FVC and/or > 15% fall in DLCO at 12 months after baseline (0.48 vs. 0.33; p = 0.023. Conclusions These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.

FcGammaRIIa polymorphism and anti-malaria specific IgG and IgG subclass responses in populations differing in susceptibility to malaria in Burkina Faso

DEFF Research Database (Denmark)

Cherif, Mariama K; Sanou, Guillaume S; Maiga, Boubakar

2012-01-01

Fc¿RIIa is known to be polymorphic; and certain variants err associated with differt susceptibilities to malaria. Studies involving the Fulani ethnic group reported an ethnic difference in Fc¿RIIa-R131H genotype frequencies between the Fulani and other sympatric groups. No previous studies have a...

Increase of lymphocytes with Fc receptors for IgE in patients with allergic rhinitis during the grass pollen season.

OpenAIRE

Spiegelberg, H L; Simon, R A

1981-01-01

Peripheral blood lymphocytes from 10 nonallergic donors and 7 patients suffering from seasonal allergic rhinitis and receiving desensitization therapy were analyzed by rosette assays for Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) before, during and after the grass pollen season. Six of seven patients had moderately elevated IgE levels (330 +/- 268 IU/ml), all had high titers of skin sensitizing antibodies to grass pollens and serum IgE antibodies as measured by radio-allergosorb...

FcεRI γ-Chain Negatively Modulates Dectin-1 Responses in Dendritic Cells

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Yi-Gen Pan

2017-10-01

Full Text Available The inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM-containing adapters DAP12 and FcεRI γ-chain (FcRγ has been found in many immune functions. Herein, we have further explored the role of these adapters in C-type lectin receptors response. We identified that FcRγ, but not DAP12, could negatively regulate the Dectin-1 responses in dendritic cells (DCs. Loss of FcRγ or both DAP12 and FcRγ enhanced the maturation and cytokine production in DCs upon Dectin-1 activation compared to normal cells, whereas DCs lacking only DAP12 showed little changes. In addition, increments of T cell activation and T helper 17 polarization induced by FcRγ-deficient DCs were observed both in vitro and in vivo. Examining the Dectin-1 signaling, we revealed that the activations of several signaling molecules were augmented in FcRγ-deficient DCs stimulated with Dectin-1 ligands. Furthermore, we demonstrated that the association of phosphatases SHP-1 and PTEN with FcRγ may contribute to the negative regulation of FcRγ in Dectin-1 activation in DCs. These results extend the inhibitory effect of ITAM-containing adapters to Dectin-1 response in immune functions, even though Dectin-1 contains an ITAM-like intracellular domain. According to the role of Dectin-1 in responding to microbes and tumor cells, our finding may have applications in the development of vaccine and cancer therapy.

A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

International Nuclear Information System (INIS)

Haas, G.G. Jr.; D'Cruz, O.J.

1989-01-01

Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

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Avery, Lindsay B.; Wang, Mengmeng; Kavosi, Mania S.; Joyce, Alison; Kurz, Jeffrey C.; Fan, Yao-Yun; Dowty, Martin E.; Zhang, Minlei; Zhang, Yiqun; Cheng, Aili; Hua, Fei; Jones, Hannah M.; Neubert, Hendrik; Polzer, Robert J.; O'Hara, Denise M.

2016-01-01

ABSTRACT Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r2 = 0.83, r = 0.91, p PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic. PMID:27232760

Multiple Plasmodium falciparum erythrocyte membrane protein 1 variants per genome can bind IgM via its Fc fragment Fcμ

DEFF Research Database (Denmark)

Jeppesen, Anine; Ditlev, Sisse Bolm; Soroka, Vladyslav

2015-01-01

with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fcμ) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report...... resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBLε domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc...

Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

NARCIS (Netherlands)

van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

1986-01-01

Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophagoïdes pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

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De Nardis, Camilla; Hendriks, Linda J A; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B H; de Kruif, John

2017-09-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Production and characterization of anti-human IgG F(ab')2 antibody fragment.

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Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

2018-04-10

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

NARCIS (Netherlands)

Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

2009-01-01

Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor FcγRIIB expression on B cells

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Duarte Alberto JS

2010-03-01

Full Text Available Abstract Background Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1

DEFF Research Database (Denmark)

Nexø, Bjørn Andersen; Christensen, Tove; Frederiksen, Jette

2011-01-01

or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We...... conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis....

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

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Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

2017-09-01

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

Hematopoietic properties of granulocyte colony-stimulating factor/immunoglobulin (G-CSF/IgG-Fc fusion proteins in normal and neutropenic rodents.

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George N Cox

Full Text Available Previously we showed that granulocyte colony-stimulating factor (G-CSF in vitro bioactivity is preserved when the protein is joined via a flexible 7 amino acid linker to an immunoglobulin-1 (IgG1-Fc domain and that the G-CSF/IgG1-Fc fusion protein possessed a longer circulating half-life and improved hematopoietic properties compared to G-CSF in normal rats. We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG -modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5 when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.

Construction and expression of an anti-VEGFR2 Nanobody-Fc fusionbody in NS0 host cell.

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Qasemi, Maryam; Behdani, Mahdi; Shokrgozar, Mohammad Ali; Molla-Kazemiha, Vahid; Mohseni-Kuchesfahani, Homa; Habibi-Anbouhi, Mahdi

2016-07-01

Angiogenesis is the formation of new blood vessels which is involved in migration, growth and differentiation of endothelial cells. This process regularly occurs during growth and development in children however, in adults is usually part of a disease process such as cancer. The vascular endothelial growth factor (VEGF) is a vital player in the vascular development and angiogenesis in physiological and pathological processes. Camelid's immune system has unique antibodies which are composed of only a heavy chain homodimer and the variable domain (VHH, Nanobody). Nanobodies are small, around 15 kDa and stable. In this study, we engineered and constructed a new Nanobody-Fc fusion protein (fusionbody) composed of an anti-VEGFR2 Nanobody and an Fc fragment of human IgG1 antibody. The recombinant vector was transfected into NS0 host cells. Stable producer clones were developed and the recombinant fusionbody was expressed and purified. Functional assay showed the anti-VEGFR2 fusionbody could bind to VEGFR2 on cell surface via VHH part and could mediate killing the targeted cells through direct cell death and complement-dependent cytotoxicity (CDC). Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

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Priscila de Faria-Pinto

2010-07-01

Full Text Available In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80% of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14 after treatment (AT with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

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Bingchun Zhao

2018-05-01

Full Text Available Epstein–Barr virus (EBV was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications.

[PHEMA/PEI]–Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma

Energy Technology Data Exchange (ETDEWEB)

Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Elkak, Assem [Laboratory of “Valorisation des Ressources Naturelles et Produits de Santé (VRNPS)”, Doctoral School of Sciences and Technology, Lebanese University, Rafic Hariri University Campus, Hadath (Lebanon); Denizli, Adil, E-mail: denizli@hacettepe.edu [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

2016-04-01

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]–Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH 7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]–Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). - Highlights: • Cu(II)-ions were coupled to PHEMA using PEI as the chelating ligand. • Cu(II) chelated [PHEMA/PEI] cryogels for IgG separation were produced. • Maximum IgG adsorption capacity

[PHEMA/PEI]–Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma

International Nuclear Information System (INIS)

Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

2016-01-01

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]–Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH 7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]–Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). - Highlights: • Cu(II)-ions were coupled to PHEMA using PEI as the chelating ligand. • Cu(II) chelated [PHEMA/PEI] cryogels for IgG separation were produced. • Maximum IgG adsorption capacity

Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay Los receptores Fc para inmunoglobulinas en cepas clínicas de Staphylococcus aureus no confieren resistencia a la fagocitosis in vitro

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Benito VEGA

1999-05-01

Full Text Available Staphylococcus aureus binds Immunoglobulin G (IgG on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01; but the former were killed more efficiently than the latter (80-90% and 40%, respectively. It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.Staphylococcus aureus liga inmunoglobulinas G (IgG a su superficie externa debido a la presencia de receptores para el dominio Fc de esas inmunoglobulinas. Este mecanismo representa una clase de camuflage contra células fagocíticas. Para confirmar tal posibilidad se realizó una evaluación in vitro de la actividad fagocítica de leucocitos polimorfonucleares (PMN contra cepas de Staphylococcus aureus, comparando 18 cepas aisladas de casos clínicos y 16 de individuos sanos. La presencia de receptores fue evaluada por hemaglutinación (HA con eritrocitos grupo A luego que las cepas fueron incubadas con IgG anti grupo sanguíneo A. La fagocitosis de S. aureus fue realizada mezclando células vivas con una suspensión de PMN e incubada a 37 °C por una hora; las bacterias sobrevivientes

Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

National Research Council Canada - National Science Library

Palys, Thomas J; Schmid, Kara E; Scherer, John M; Schoepp, Randal J

2006-01-01

.... Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells...

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

Science.gov (United States)

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults

OpenAIRE

Gaudinski, Martin R.; Coates, Emily E.; Houser, Katherine V.; Chen, Grace L.; Yamshchikov, Galina; Saunders, Jamie G.; Holman, LaSonji A.; Gordon, Ingelise; Plummer, Sarah; Hendel, Cynthia S.; Conan-Cibotti, Michelle; Lorenzo, Margarita Gomez; Sitar, Sandra; Carlton, Kevin; Laurencot, Carolyn

2018-01-01

Background VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor. Methods and findings This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccin...

Hydrodynamic delivery of plasmid DNA encoding human FcγR-Ig dimers blocks immune-complex mediated inflammation in mice.

Science.gov (United States)

Shashidharamurthy, R; Machiah, D; Bozeman, E N; Srivatsan, S; Patel, J; Cho, A; Jacob, J; Selvaraj, P

2012-09-01

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment.

Science.gov (United States)

Chan, Anita S Y; Mudhar, Hardeep; Shen, Sunny Yu; Lang, Stephanie S; Fernando, Malee; Hilmy, Maryam Hazly; Guppy, Naomi Jayne; Rennie, Ian; Dunkley, Lisa; Al Jajeh, Issam

2017-11-01

To determine the role of serum and tissue IgG2 in orbital biopsies with the histological features of IgG4-related disease (IgG4-RD) in comparison with non-IgG4-related orbital inflammatory disorders (OID), including autoimmune disorders. This is an international (Sheffield, UK, and Singapore) collaborative, retrospective case review of 69 patients (38 from Singapore National Eye Centre and 31 from Royal Hallamshire Hospital, Sheffield) with orbital inflammatory biopsies between 2002 and 2016. Clinical information and histology were reviewed and cases were classified into three groups: Group 1: IgG4-RD orbital inflammation (n=43); Group 2: idiopathic OID (n=12) and Group 3: autoimmune OID (n=14). Serum IgG1, IgG2, IgG3 and IgG4 levels were collated where available and immunohistochemistry (IHC) for tissue IgG2 plasma cells was performed. Dual IHC showed IgG2 plasma cells as a distinct population from IgG4 plasma cells. Significant (twofold) serum IgG2 elevation was noted among IgG4-RD (group 1), idiopathic (group 2) and autoimmune OID (group 3). Similarly, significant elevation of tissue IgG2 plasma cells was also seen among IgG4-RD (group 1), idiopathic and autoimmune OID (groups 2 and 3). Significant elevations of serum IgG2 and tissue IgG2 plasma cells are present in orbital IgG4-RD in comparison with non-IgG4 orbital inflammation (idiopathic and autoimmune OID), suggesting that IgG2 may play a role in IgG4-RD. A serum IgG2 cut-off >5.3 g/L was found to be 80% sensitive and 91.7% specific for orbital IgG4-RD, with an accuracy of 0.90. Tissue IgG2 and IgG4 subclass reporting may provide additional insight regarding the 'IgG4-RD' pathogenesis. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Science.gov (United States)

Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

2013-01-01

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

Science.gov (United States)

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Pathogenicity of IgG in patients with IgG4-related disease.

Science.gov (United States)

Shiokawa, Masahiro; Kodama, Yuzo; Kuriyama, Katsutoshi; Yoshimura, Kenichi; Tomono, Teruko; Morita, Toshihiro; Kakiuchi, Nobuyuki; Matsumori, Tomoaki; Mima, Atsushi; Nishikawa, Yoshihiro; Ueda, Tatsuki; Tsuda, Motoyuki; Yamauchi, Yuki; Minami, Ryuki; Sakuma, Yojiro; Ota, Yuji; Maruno, Takahisa; Kurita, Akira; Sawai, Yugo; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumura, Kazuyoshi; Watanabe, Tomohiro; Notohara, Kenji; Tsuruyama, Tatsuaki; Seno, Hiroshi; Chiba, Tsutomu

2016-08-01

IgG4-related disease (IgG4-RD) is a systemic disease characterised by elevated serum IgG4 and IgG4-positive lymphoplasmacytic infiltration in the affected tissues. The pathogenic role of IgGs, including IgG4, in patients with IgG4-RD, however, is unknown. We examined the pathogenic activity of circulating IgGs in patients with IgG4-RD by injecting their IgGs into neonatal male Balb/c mice. Binding of patient IgGs to pancreatic tissue was also analysed in an ex vivo mouse organ culture model and in tissue samples from patients with autoimmune pancreatitis (AIP). Subcutaneous injection of patient IgG, but not control IgG, resulted in pancreatic and salivary gland injuries. Pancreatic injury was also induced by injecting patient IgG1 or IgG4, with more destructive changes induced by IgG1 than by IgG4. The potent pathogenic activity of patient IgG1 was significantly inhibited by simultaneous injection of patient IgG4. Binding of patient IgG, especially IgG1 and IgG4, to pancreatic tissue was confirmed in both the mouse model and AIP tissue samples. IgG1 and IgG4 from patients with IgG4-RD have pathogenic activities through binding affected tissues in neonatal mice. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

Science.gov (United States)

Qureshi, O S; Rowley, T F; Junker, F; Peters, S J; Crilly, S; Compson, J; Eddleston, A; Björkelund, H; Greenslade, K; Parkinson, M; Davies, N L; Griffin, R; Pither, T L; Cain, K; Christodoulou, L; Staelens, L; Ward, E; Tibbitts, J; Kiessling, A; Smith, B; Brennan, F R; Malmqvist, M; Fallah-Arani, F; Humphreys, D P

2017-12-06

Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

DEFF Research Database (Denmark)

van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.

2015-01-01

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexameri...

Effects of sterilizing radiation dose on the amino acid composition of normal human Ig(G)

International Nuclear Information System (INIS)

Kaupert, N.L.; Mariano, E.E.

1981-01-01

In order to verify gamma radiation effect of 60 Co, samples of Normal human Ig(G) in a) pH 7 solutions with different concentrations, and b) in liophilized state, were irradiated. In both, the quali and quantitative amino acid compositions have been studied. No changes in amino acid composition were observed, for doses up to 10 Mrad delivered to the liophilized samples. Nevertheless, when 5 mg/ml and 10 mg/ml solutions of Ig(G) were irradiated with a 2 Mrad gamma dose, both were affected. The damage appeared in greater proportion in the samples with lower concentration. Cistine was the amino acid most damaged and the loss of methionine, proline histidine, arginine, tirosine and phenilalanine, decreased in this order. The analysis of the experimental data shows that liophilized human Ig(G) can be treated with doses higher than those required to achieve sterilization, without modifying its immunologic and primary proteic structure properties. Therefore, gamma sterilization feasibility has been proved for normal human Ig(G) only in the liophilized state. (author) [es

Pattern of pre-existing IgG subclass responses to a panel of asexual stage malaria antigens reported during the lengthy dry season in Daraweesh, Sudan

DEFF Research Database (Denmark)

Nasr, A; Iriemenam, N C; Troye-Blomberg, M

2011-01-01

The anti-malarial IgG immune response during the lengthy and dry season in areas of low malaria transmission as in Eastern Sudan is largely unknown. In this study, ELISA was used for the measurement of pre-existing total IgG and IgG subclasses to a panel of malaria antigens, MSP2-3D7, MSP2-FC27, ...

Attenuated atherosclerotic lesions in apoe-fc gamma-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

Science.gov (United States)

Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for Fc gamma Rs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating Fc gamma R in the progressio...

Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

International Nuclear Information System (INIS)

Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

1980-01-01

A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively

Preclinical evaluation of VIS513, a therapeutic antibody against dengue virus, in non-human primates.

Science.gov (United States)

Ong, Eugenia Z; Budigi, Yadunanda; Tan, Hwee Cheng; Robinson, Luke N; Rowley, Kirk J; Winnett, Alexander; Hobbie, Sven; Shriver, Zachary; Babcock, Gregory J; Ooi, Eng Eong

2017-08-01

Despite useful in vivo activity, no therapeutic against dengue virus (DENV) has demonstrated efficacy in clinical trials. Herein, we explored dosing and virological endpoints to guide the design of human trials of VIS513, a pan-serotype anti-DENV IgG1 antibody, in non-human primates (NHPs). Dosing VIS513 pre- or post-peak viremia in NHPs neutralized infectious DENV although RNAemia remained detectable post-treatment; differential interaction of human IgGs with macaque Fc-gamma receptors may delay clearance of neutralized DENV. Our findings suggest useful antiviral utility of VIS513 and highlight an important consideration when evaluating virological endpoints of trials for anti-DENV biologics. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

Science.gov (United States)

Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

2017-09-29

Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

Science.gov (United States)

Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

2014-12-01

Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

Homogeneous immunoassay for human IgG using oriented hen egg IgY immobilized on gold sol nanoparticles

International Nuclear Information System (INIS)

Yeritsyan, H.E.; Gasparyan, V.K.

2012-01-01

Homogeneous immunoassays using (red) gold nanoparticles represent an attractive detection scheme because of the option of photometric readout. We have applied oriented immobilization of hen egg immunoglobulin Y (IgY) on gold nanoparticles when developing a homogeneous immunoassay for human IgG. In oriented immobilization, as opposed to random immobilization, the antigen binding capabilities of the antibodies are retained. It is shown that such immunoassay has significantly better sensitivity in comparison with methods based on conventional immobilization of affinity-purified antibodies. It is also shown that hen egg IgY is better suited than rabbit antibodies, because much more antibody can be immobilized on gold nanoparticles without any destabilization, probably because of the more acidic nature of these antibodies. In addition, hen egg IgY can be supplied in higher quantity and can be prepared more easily than IgG from rabbits. Bleeding and slaughtering of animals is not needed. The assay presented here has a wide detection range (30-500 ng. mL -1 ) and a limit of detection as low as 30 ng. mL -1 of human IgG. (author)

Close-up of the alpha-1,3-Gal epitope as defined by a monoclonal chimeric IgE and human serum using saturation transfer difference (STD) NMR

DEFF Research Database (Denmark)

Plum, Melanie; Michel, Yvonne; Wallach, Katharina

2011-01-01

of an alpha-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed...... by mediator release assays, surface plasmon resonance (SPR) and STD NMR analyses. The alpha-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference...

A rapid radioassay for human IgG produced in lymphocyte in vitro culture systems

International Nuclear Information System (INIS)

Weightman, D.R.; Shale, D.J.; Tomlinson, W.R.

1981-01-01

Protein A bearing Staphylococcus aureus was used to develop a solid-phase radioassay for IgG immunoglobulins. The assay was specifically optimised for use in vitro human lymphocyte culture work. Compared with a solid-phase radioimmunoassay for IgG produced in lymphocyte culture, this assay had a similar performance profile and the advantages of rapidity and technical ease. (Auth.)

Anti-influenza Hyperimmune Immunoglobulin Enhances Fc-functional Antibody Immunity during Human Influenza Infection.

Science.gov (United States)

Vanderven, Hillary A; Wragg, Kathleen; Ana-Sosa-Batiz, Fernanda; Kristensen, Anne B; Jegaskanda, Sinthujan; Wheatley, Adam K; Wentworth, Deborah; Wines, Bruce D; Hogarth, P Mark; Rockman, Steve; Kent, Stephen J

2018-05-31

New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralising antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fc receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1-3 days against infecting and non-infecting strains of influenza. Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralising antibodies in the Flu-IVIG preparation.

Hydrodynamic delivery of plasmid DNA encoding human Fc?R-Ig dimers blocks immune-complex mediated inflammation in mice

OpenAIRE

Shashidharamurthy, Rangaiah; Machiah, Deepa; Bozeman, Erica N.; Srivatsan, Sanjay; Patel, Jaina; Cho, Alice; Jacob, Joshy; Selvaraj, Periasamy

2011-01-01

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcgamma receptor ?Ig fusion molecules (Fc?R-Igs) in mice by administering Fc?R-Ig plasmid DNAs hydrodynamically and compared their effectiveness to purified molecules in blocking immune-complex (IC) mediated inflammation in mice. The concentration of hydrodynamically expressed Fc?R-Igs (CD16AF-Ig, CD32AR-Ig and CD32AH-Ig) reached a maximum of ...

Detection of acute inflammation with 111In-labeled nonspecific polyclonal IgG

International Nuclear Information System (INIS)

Fischman, A.J.; Rubin, R.H.; Khaw, B.A.

1988-01-01

The detection of focal sites of inflammation is an integral part of the clinical evaluation of the febrile patient. When anatomically distinct abscesses are present, lesion detection can be accomplished by standard radiographic techniques, particularly in patients with normal anatomy. At the phlegmon stage, however, and in patients who have undergone surgery, these techniques are considerably less effective. While radionuclide methods, such as Gallium-67 (67Ga)-citrate and Indium-111 (111In)-labeled WBCs have been relatively successful for the detection of early inflammation, neither approach is ideal. In the course of studies addressing the use of specific organism-directed antibodies for imaging experimental infections in animals, we observed that nonspecific polyclonal immunoglobulin G (IgG) localized as well as specific antibodies. Preliminary experiments suggested that the Fc portion of IgG is necessary for effective inflammation localization. Since polyclonal IgG in gram quantities has been safely used for therapy in patients with immune deficiency states, we decided to test whether milligram quantities of radiolabeled IgG could image focal sites of inflammation in humans. Thus far, we have studied a series of 84 patients with suspected lesions in the abdomen, pelvis, vascular grafts, lungs, or bones/joints. In 48 of 52 patients with focal lesions detected by surgery, computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), the IgG scan correctly localized the site, while 31 patients without focal inflammation had no abnormal focal localization of the radiopharmaceutical. Four patients had false negative scans and one patient had a false positive scan. For this small series, the overall sensitivity and specificity were 92% and 95%, respectively. In this report, we review our experience with this exciting new agent

Dicty_cDB: FC-BR21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BR21 (Link to dictyBase) - - - Contig-U15384-1 | Contig-U16443-1 FC-BR2...1P (Link to Original site) FC-BR21F 551 FC-BR21Z 122 FC-BR21P 673 - - Show FC-BR21 Library FC (L...ink to library) Clone ID FC-BR21 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Cont...ig-U15384-1 | Contig-U16443-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BR/FC-BR2...1Q.Seq.d/ Representative seq. ID FC-BR21P (Link to Original site) Representative DNA sequence >FC-BR21 (FC-BR2

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

Science.gov (United States)

Soleimanpour, Saman; Farsiani, Hadi; Mosavat, Arman; Ghazvini, Kiarash; Eydgahi, Mohammad Reza Akbari; Sankian, Mojtaba; Sadeghian, Hamid; Meshkat, Zahra; Rezaee, Seyed Abdolrahim

2015-12-01

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.

IgG and IgG subclasses antibody responses to rK39 in Leishmania donovani infections

International Nuclear Information System (INIS)

Daifalla, N.S.; El Hassan, A.M.

1998-01-01

Leishmania donovani infection cause a wide spectrum of human diseases ranging from self-healing subclinical infections to severe visceral leishmaniasis, post kal-azar dermal leishmaiasis, and mucosal leishmaiasis. The infection associated with high levels of anti-leishmania antibodies which offer a potential parameter for the serological diagnosis of L. donovani infection replacing the invasive parasitological methods. rK39, a cloned antigen of L. chagasis was reported to have high levels of anti-leishmania antibodies in Sudanese and American visceral leishmaniasis patients. In an assessment of rK39-ELISA in detecting L. donovani infection we found that the antigen detected visceral leishmaniasis, post kala-azar dermal leishmaniasis, and mucosal leismaniasis with the sensitives of 96.6%, 95.91% and 90.91% respectively. The test has the specificity of 96.7%. Further investigation of 25 visceral leishmaniasis patients showed elevated anti-rK39 antibody responses of IgG subclasses with IgG1 and IgG3 significantly higher than IgG4. igG3 showed the highest sensitivity (84.00%) whereas IgG1 showed the highest sensitivity (100%). The dynamics of the serological reactivity to rK39 in l.donovani infections will be discussed in relation to exposure, infection, cure and relapse.(Author)

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

Science.gov (United States)

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies

International Nuclear Information System (INIS)

Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J.

1990-01-01

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab

Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles.

Science.gov (United States)

Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T; Tsourkas, Andrew

2014-08-27

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Envelope proteins of bovine herpesvirus 1: immunological and biochemical studies

International Nuclear Information System (INIS)

Rodriguez Roque, L.L.

1986-01-01

The authors studied immunological and biochemical properties of the bovid herpesvirus 1 (BHV-1) envelope proteins in order to understand the pathogenesis of BHV-1 infection and to provide basic information for the production of effective subunit vaccines against BHV-1. Ten glycoproteins MW 180, 150, 130, 115, 97, 77, 74, 64, 55, and 45 kilodaltons (K), and a single non-glycosylated 108 K protein were quantitatively removed from purified BHV-1 virions by detergent treatment. These glycoproteins were present on the virion envelope and on the surface of BHV-1 infected cells. The quantitative removal from virions by treatment with nonionic detergents and their presence on the surface of infected cells indicate that 180/97, 150/77, and 130/74/55 K are major components of the BHV-1 envelope and are also the targets of virus neutralizing humoral immune response. Envelope glycoproteins of herpes simplex type 1 (HSV-1) bind immunoglobulin by the Fc end and it is suggested this may increase pathogenicity of this virus. They searched for a similar function in BVH-1 by measuring the ability of BHV-1 infected cells and viral envelope proteins to bind radiolabelled rabbit and bovine IgG. Binding activity for rabbit IgG or bovine IgG-Fc could not be demonstrated by BHV-1 infected MDBK cells, whereas, MDBK cells infected with HSV-1 bound rabbit IgG and bovine IgG-Fc. None of the three major envelope proteins of BHV-1 bound to rabbit or bovine IgG. The results of this study indicate that BHV-1, unlike some other herpesviruses, lack Fc binding activity

The effect of FcγRIIA and FcγRIIB on coronary artery lesion formation and intravenous immunoglobulin treatment responses in children with Kawasaki disease

Science.gov (United States)

Chang, Ling-Sai; Lo, Mao-Hung; Li, Sung-Chou; Yang, Ming-Yu; Hsieh, Kai-Sheng; Kuo, Ho-Chang

2017-01-01

Previous research has found patients with the FcγRIIIB NA1 variant having increased risk of intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD). Our previous studies revealed that elevated FcγRIIA expression correlated with the susceptibility of KD patients. We conducted this research to determine whether and how Fcγ receptors affect the susceptibility, IVIG treatment response, and coronary artery lesions (CAL) of KD patients. The activating FcγRIIA and inhibitory FcγRIIB methylation levels of seven patients with KD and four control subjects were examined using HumanMethylation27 BeadChip. We enrolled a total of 44 KD patients and 10 control subjects with fevers. We performed real-time RT-PCR to determine the FcγRIIA and FcγRIIB expression levels, as well as a luciferase assay of FcγRIIA. We found a considerable increase in methylation of both FcγRIIA and FcγRIIB in KD patients undergoing IVIG treatment. Promoter methylation of FcγRIIA inhibited reporter activity in K562 cells using luciferase assay. The FcγRIIB mRNA expression levels were not found to increase susceptibility, CAL formation, or IVIG resistance. FcγRIIA mRNA expression levels were significantly higher in IVIG-resistant patients than in those that responded to IVIG during the pre-treatment period. Furthermore, the FcγRIIA/IIB mRNA expression ratio was considerably higher in KD patients with CAL than in those without CAL. FcγRIIA and FcγRIIB both demonstrated increased methylation levels in KD patients that underwent IVIG treatment. FcγRIIA expression influenced the IVIG treatment response of KD patients. The FcγRIIA/IIB mRNA expression ratio was greater in KD patients with CAL formation. PMID:27893416

Dicty_cDB: FC-AI07 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI07 (Link to dictyBase) - - - Contig-U15296-1 | Contig-U15756-1 FC-AI...07P (Link to Original site) FC-AI07F 580 FC-AI07Z 723 FC-AI07P 1303 - - Show FC-AI07 Library FC (...Link to library) Clone ID FC-AI07 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15296-1 | Contig-U15756-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...07Q.Seq.d/ Representative seq. ID FC-AI07P (Link to Original site) Representative DNA sequence >FC-AI07 (FC-AI

Dicty_cDB: FC-AI22 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI22 (Link to dictyBase) - - - Contig-U15369-1 | Contig-U15732-1 FC-AI...22P (Link to Original site) FC-AI22F 583 FC-AI22Z 683 FC-AI22P 1266 - - Show FC-AI22 Library FC (...Link to library) Clone ID FC-AI22 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15369-1 | Contig-U15732-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...22Q.Seq.d/ Representative seq. ID FC-AI22P (Link to Original site) Representative DNA sequence >FC-AI22 (FC-AI

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

Science.gov (United States)

Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

2009-11-17

The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Natural IgG autoantibodies are abundant and ubiquitous in human sera, and their number is influenced by age, gender, and disease.

Directory of Open Access Journals (Sweden)

Eric P Nagele

Full Text Available The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer's and Parkinson's diseases.

Highly sensitive electrochemical immunoassay for human IgG using double-encoded magnetic redox-active nanoparticles

International Nuclear Information System (INIS)

Tang, D.; Tang, J.; Su, B.; Chen, H.; Chen, G.; Huang, J.

2010-01-01

A new sandwich-type electrochemical immunoassay was developed for the detection of human IgG using doubly-encoded and magnetic redox-active nanoparticles as recognition elements on the surface of a glassy carbon electrode modified with anti-IgG on nanogold particles. The recognition elements were synthesized by coating magnetic Fe3O4 nanoparticles with Prussian blue nanoparticles and then covered with peroxidase-labeled anti-IgG antibodies (POx-anti-IgG) on Prussian blue nanoparticles. The immunoelectrode displays very good electrochemical properties towards detection of IgG via using double-encoded magnetic redox-active nanoparticles as trace and hydrogen peroxide as enzyme substrate. Its limit of detection (10 pmol.L -1 ) is 10-fold better than that of using plain POx-anti-IgG secondary antibodies. The method was applied to the detection of IgG in serum samples, and an excellent correspondence with the reference values was found. (author)

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

Science.gov (United States)

Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

2012-10-15

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

Dicty_cDB: FC-AI15 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI15 (Link to dictyBase) - G01730 DDB0214993 Contig-U15123-1 FC-AI...15E (Link to Original site) - - - - - - FC-AI15E 856 Show FC-AI15 Library FC (Link to library) Clone ID FC-AI...3-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI15Q.Se...q.d/ Representative seq. ID FC-AI15E (Link to Original site) Representative DNA sequence >FC-AI15 (FC-AI15Q) /CSM/FC/FC-AI/FC-AI...AAAAAAAATA sequence update 1996.12.24 Translated Amino Acid sequence kt*riyi*KMMIKYITIAILFIASLVKADLQFSLCPTCV

A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

Science.gov (United States)

Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

2014-07-01

The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection. Copyright © 2014 Elsevier Inc. All rights reserved.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

OpenAIRE

Song, Yuanda; Dilger, Emily; Bell, Jessica; Barton, William A; Fang, Xianjun

2010-01-01

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29–915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated fro...

Fc-based delivery system enhances immunogenicity of a tuberculosis subunit vaccine candidate consisting of the ESAT-6:CFP-10 complex.

Science.gov (United States)

Farsiani, Hadi; Mosavat, Arman; Soleimanpour, Saman; Sadeghian, Hamid; Akbari Eydgahi, Mohammad Reza; Ghazvini, Kiarash; Sankian, Mojtaba; Aryan, Ehsan; Jamehdar, Saeid Amel; Rezaee, Seyed Abdolrahim

2016-06-21

Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guérin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.

Dicty_cDB: FC-AI08 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI08 (Link to dictyBase) - G01729 DDB0233148 Contig-U14939-1 FC-AI...08P (Link to Original site) FC-AI08F 654 FC-AI08Z 563 FC-AI08P 1217 - - Show FC-AI08 Library FC (Link... to library) Clone ID FC-AI08 (Link to dictyBase) Atlas ID - NBRP ID G01729 dictyBase ID DDB0233148 Link to ...Contig Contig-U14939-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...08Q.Seq.d/ Representative seq. ID FC-AI08P (Link to Original site) Representative DNA sequence >FC-AI08 (FC-AI

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

International Nuclear Information System (INIS)

Rieber, E.P.; Linke, R.P.; Riethmueller, G.; Heyden, H.W. von; Waller, H.D.

1976-01-01

Using 125 I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab') 2 -fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of μ-chains was detected. γ-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria. (orig.) [de

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

Energy Technology Data Exchange (ETDEWEB)

Rieber, E P; Linke, R P; Riethmueller, G [Tuebingen Univ. (Germany, F.R.). Abt. fuer Experimentelle Chirurgie und Immunologie; Heyden, H.W. von; Waller, H D [Tuebingen Univ. (Germany, F.R.). Abt. Innere Medizin 2

1976-01-01

Using /sup 125/I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')/sub 2/-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of ..mu..-chains was detected. ..gamma..-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

Science.gov (United States)

Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

Directory of Open Access Journals (Sweden)

Masaki Kurogochi

Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

OpenAIRE

Kontny, U; Kurane, I; Ennis, F A

1988-01-01

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

Neisseria meningitidis Group A IgG1 and IgG2 Subclass Immune Response in African Children Aged 12–23 Months Following Meningococcal Vaccination

Science.gov (United States)

Holme, Daniel; Findlow, Helen; Sow, Samba O.; Idoko, Olubukola T.; Preziosi, Marie-Pierre; Carlone, George; Plikaytis, Brian D.; Borrow, Ray

2015-01-01

Background. A group A meningococcal conjugate vaccine, PsA-TT, was licensed in 2010 and was previously studied in a phase 2 clinical trial to evaluate its safety and immunogenicity in African children 12–23 months of age. Methods. Subjects received either PsA-TT; meningococcal group A, C, W, Y polysaccharide vaccine (PsACWY); or Haemophilus influenzae type b conjugate vaccine (Hib-TT). Forty weeks following primary vaccination, the 3 groups were further randomized to receive either PsA-TT, one-fifth dose of PsACWY, or Hib-TT. Group A–specific immunoglobulin G (IgG) subclass response was characterized using an enzyme-linked immunosorbent assay. Results. The predominant IgG subclass response, regardless of vaccine, was IgG1. One month following primary vaccination, the geometric mean concentrations (GMCs) of IgG1 and IgG2 in the PsA-TT group were 21.73 µg/mL and 6.27 µg/mL, whereas in the PsACWY group the mean GMCs were 2.01 µg/mL and 0.97 µg/mL, respectively (P Group A–specific IgG1 and IgG2 GMCs remained greater in the PsA-TT group than in the PsACWY group 40 weeks following primary vaccination (P vaccines. Conclusions. Vaccination of African children aged 12–24 months with either PsA-TT or PsACWY elicited a predominantly IgG1 response. The IgG1:IgG2 mean ratio decreased following successive vaccination with PsACWY, indicating a shift toward IgG2, suggestive of the T-cell–independent immune response commonly associated with polysaccharide antigens. Clinical Trials Registration. SRCTN78147026. PMID:26553689

Anticomplementary Activity of Horse IgG and F(ab')2 Antivenoms

OpenAIRE

Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.

2014-01-01

2094-01 Embargo por política editorial Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several b...

External apical root resorption diagnosis by using FII human dentine fraction and salivary IGg.

Science.gov (United States)

Da-Costa, Tânia Maris Pedrini Soares; Hidalgo, Mirian Marubayashi; Consolaro, Alberto; Lima, Carlos Eduardo de Oliveira; Tanaka, Evelise Ono; Itano, Eiko Nakagawa

2018-06-01

External apical root resorption as a consequence of orthodontic treatment is an inflammatory pathological process that results in permanent loss of tooth structure from the root apex. This study aimed to investigate the diagnostic potential of human dentine fractions and salivary IgG in external apical root resorption. Saliva samples were collected from 10 patients before (T0) and after 3 (T3), 6 (T6) and 12 (T12) months of orthodontic treatment. The total dentinal extract, obtained from human third molars, was fractioned by gel filtration chromatography in three fractions denominated FI, FII and FIII. The root resorption analysis of the upper central incisors was performed by digital image subtraction method. Reactivity of salivary IgG to antigenic fractions of dentine was determined by enzyme-linked immunosorbent assay (Elisa). Regardless of treatment, FI dentin fraction with high MM (root resorptions were detected. Our results suggest that FII human dentine fraction and salivary IgG have potential to be used in diagnosis and monitoring of external apical root resorption. The development of a practical and accessible biochemical test using saliva and FII dentine fraction may help in the prevention of severe root resorption. Copyright © 2018. Published by Elsevier Masson SAS.

Dicty_cDB: FC-BS11 [Dicty_cDB

Lifescience Database Archive (English)

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FcγRIIb on myeloid cells rather than on B cells protects from collagen-induced arthritis.

Science.gov (United States)

Yilmaz-Elis, A Seda; Ramirez, Javier Martin; Asmawidjaja, Patrick; van der Kaa, Jos; Mus, Anne-Marie; Brem, Maarten D; Claassens, Jill W C; Breukel, Cor; Brouwers, Conny; Mangsbo, Sara M; Boross, Peter; Lubberts, Erik; Verbeek, J Sjef

2014-06-15

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type-specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell-specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell-specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility. Copyright © 2014 by The American Association of Immunologists, Inc.

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain*

Science.gov (United States)

Vaughan, Andrew T.; Chan, Claude H. T.; Klein, Christian; Glennie, Martin J.; Beers, Stephen A.; Cragg, Mark S.

2015-01-01

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. PMID:25568316

Dicty_cDB: FC-AY04 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI04 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI10 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI10 (Link to dictyBase) - - - Contig-U16270-1 FC-AI10F (Li...nk to Original site) FC-AI10F 405 - - - - - - Show FC-AI10 Library FC (Link to library) Clone ID FC-AI10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI10Q.Seq.d/ Representative seq. ID FC-AI...10F (Link to Original site) Representative DNA sequence >FC-AI10 (FC-AI10Q) /CSM/FC/FC-AI/FC-AI10Q.Seq.... sequence RKKRKSDYTSFSTYIHKLLKQITPPTNAKSNEKGDRKFTISSKAMSVMNSFVHDIFDRIA TEASGLAKKKKRQTLHSRDIQVAVRIILTGELAXHAI

Dicty_cDB: FC-AI23 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI23 (Link to dictyBase) - - - Contig-U15308-1 FC-AI23Z (Li...nk to Original site) - - FC-AI23Z 603 - - - - Show FC-AI23 Library FC (Link to library) Clone ID FC-AI23 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI23Q.Seq.d/ Representative seq. ID FC-AI...23Z (Link to Original site) Representative DNA sequence >FC-AI23 (FC-AI23Q) /CSM/FC/FC-AI/FC-AI23Q.Seq....LNTLAKKNEQVVEGEILAKQLTGVTAEELSEFKACFSHFDKDN DNKLNRLEFSSCLKSIGDELTEEQLNQVISKIDTDGNGTISFEEFIDYMVSSRKGTDSVE STKAAFKVMAEDKDFITEAQIRAAI

Dicty_cDB: FC-AI05 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI21 (Link to dictyBase) - - - Contig-U16254-1 FC-AI21Z (Li...nk to Original site) - - FC-AI21Z 696 - - - - Show FC-AI21 Library FC (Link to library) Clone ID FC-AI21 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI21Q.Seq.d/ Representative seq. ID FC-AI...21Z (Link to Original site) Representative DNA sequence >FC-AI21 (FC-AI21Q) /CSM/FC/FC-AI/FC-AI21Q.Seq....YPGYMYTDLSTIYERAGRIQGRNGSITQI PILTMPNDDITHPIPDLTGYITEGQIFIDRQINNRQIYPPINVLPSLSRLMKSAI

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

Science.gov (United States)

Käsermann, Fabian; Boerema, David J; Rüegsegger, Monika; Hofmann, Andreas; Wymann, Sandra; Zuercher, Adrian W; Miescher, Sylvia

2012-01-01

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG after lectin fractionation.

Directory of Open Access Journals (Sweden)

Fabian Käsermann

Full Text Available It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1 or acidified lactose (E2 were analyzed for total IgG, F(ab'(2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2; again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10% of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

Dicty_cDB: FC-BS10 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS10 (Link to dictyBase) - - - Contig-U16283-1 FC-BS10Z (Li...nk to Original site) - - FC-BS10Z 720 - - - - Show FC-BS10 Library FC (Link to library) Clone ID FC-BS10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS10Q.Seq.d/ Representative seq. ID FC-BS...10Z (Link to Original site) Representative DNA sequence >FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq....E Sequences producing significant alignments: (bits) Value FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq.d/

Dicty_cDB: FC-BS21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS21 (Link to dictyBase) - - - Contig-U15764-1 FC-BS21Z (Li...nk to Original site) - - FC-BS21Z 723 - - - - Show FC-BS21 Library FC (Link to library) Clone ID FC-BS21 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS21Q.Seq.d/ Representative seq. ID FC-BS...21Z (Link to Original site) Representative DNA sequence >FC-BS21 (FC-BS21Q) /CSM/FC/FC-BS/FC-BS21Q.Seq.... Score E Sequences producing significant alignments: (bits) Value FC-BS21 (FC-BS21Q) /CSM/FC/FC-BS/FC-BS21Q.

Dicty_cDB: FC-BS19 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI01 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-BS14 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI17 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI17 (Link to dictyBase) - - - Contig-U15690-1 FC-AI17P (Li...nk to Original site) FC-AI17F 587 FC-AI17Z 626 FC-AI17P 1212 - - Show FC-AI17 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI17Q.Seq.d/ Representative seq. ID FC-AI...17P (Link to Original site) Representative DNA sequence >FC-AI17 (FC-AI17Q) /CSM/FC/FC-AI/FC-AI...slated Amino Acid sequence ANIATVGDFLKADTVVPKMIITYNKRKQGTDYLKAVIGPILSNVIKQELNLELKPNLVYA AIISEQEIRTGEKSTLDRNV

Dicty_cDB: FC-AI18 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI12 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI19 [Dicty_cDB

Lifescience Database Archive (English)

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Dicty_cDB: FC-AI14 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI14 (Link to dictyBase) - - - Contig-U16280-1 FC-AI14Z (Li...nk to Original site) - - FC-AI14Z 671 - - - - Show FC-AI14 Library FC (Link to library) Clone ID FC-AI14 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI14Q.Seq.d/ Representative seq. ID FC-AI...14Z (Link to Original site) Representative DNA sequence >FC-AI14 (FC-AI14Q) /CSM/FC/FC-AI/FC-AI14Q.Seq....nqrllv*lvvlskklqllnsnqsfkfkkvq rmkknsvkntkn*rfvllt*nlkslkrmpksknsptkliifilkly Frame B: ---lkdl*krtphl*stcfhhptlcssrrfclwslnai

Dicty_cDB: FC-AI06 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI06 (Link to dictyBase) - - - Contig-U16465-1 FC-AI06E (Li...nk to Original site) - - - - - - FC-AI06E 1138 Show FC-AI06 Library FC (Link to library) Clone ID FC-AI06 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI06Q.Seq.d/ Representative seq. ID FC-AI...06E (Link to Original site) Representative DNA sequence >FC-AI06 (FC-AI06Q) /CSM/FC/FC-AI/FC-AI06Q.Seq...FGRGIDIERVNVVINYDMAESADTYLHRVGRAGRFGTK GLAISFVPSKEDPVLEQVQSKFVVSIKELVATPDPSTYMSG*kkkkkkkknlfvlksikk k*kkk*in

Dicty_cDB: FC-AI09 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI09 (Link to dictyBase) - - - Contig-U16149-1 FC-AI09Z (Li...nk to Original site) - - FC-AI09Z 591 - - - - Show FC-AI09 Library FC (Link to library) Clone ID FC-AI09 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI09Q.Seq.d/ Representative seq. ID FC-AI...09Z (Link to Original site) Representative DNA sequence >FC-AI09 (FC-AI09Q) /CSM/FC/FC-AI/FC-AI09Q.Seq....*tkl ik*ilifykiknnkkkkkk Frame B: ---gt*kvpeflailfkrmasrsvlwy*rcltkakkglkapqtltik

Targeting IgG in Arthritis: Disease Pathways and Therapeutic Avenues

Directory of Open Access Journals (Sweden)

Kutty Selva Nandakumar

2018-02-01

Full Text Available Rheumatoid arthritis (RA is a polygenic and multifactorial syndrome. Many complex immunological and genetic interactions are involved in the final outcome of the clinical disease. Autoantibodies (rheumatoid factors, anti-citrullinated peptide/protein antibodies are present in RA patients’ sera for a long time before the onset of clinical disease. Prior to arthritis onset, in the autoantibody response, epitope spreading, avidity maturation, and changes towards a pro-inflammatory Fc glycosylation phenotype occurs. Genetic association of epitope specific autoantibody responses and the induction of inflammation dependent and independent changes in the cartilage by pathogenic autoantibodies emphasize the crucial contribution of antibody-initiated inflammation in RA development. Targeting IgG by glyco-engineering, bacterial enzymes to specifically cleave IgG/alter N-linked Fc-glycans at Asn 297 or blocking the downstream effector pathways offers new avenues to develop novel therapeutics for arthritis treatment.

Dicty_cDB: FC-BF01 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BF01 (Link to dictyBase) - - - Contig-U15105-1 FC-BF01Z (Li...nk to Original site) - - FC-BF01Z 674 - - - - Show FC-BF01 Library FC (Link to library) Clone ID FC-BF01 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15105-1 Original site URL http://dict...402813_1( AF402813 |pid:none) Ictalurus punctatus 40S ribosomal ... 260 3e-68 AJ783868_1( AJ783868 |pid:none...lasmic 8.0 %: mitochondrial 4.0 %: nuclear >> prediction for FC-BF01 is cyt 5' end seq. ID - 5' end seq. - L

Dicty_cDB: FC-BC16 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BC16 (Link to dictyBase) - - - Contig-U15105-1 FC-BC16Z (Li...nk to Original site) - - FC-BC16Z 620 - - - - Show FC-BC16 Library FC (Link to library) Clone ID FC-BC16 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15105-1 Original site URL http://dict...4 2e-66 AY961522_1( AY961522 |pid:none) Lysiphlebus testaceipes ribosomal ... 254 2e-66 AF402813_1( AF402813 |pid:none) Ict...hondrial 4.0 %: nuclear >> prediction for FC-BC16 is cyt 5' end seq. ID - 5' end seq. - Length of 5' end seq

Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs.

Directory of Open Access Journals (Sweden)

Olivier Reynard

Full Text Available Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs

Science.gov (United States)

Reynard, Olivier; Jacquot, Frédéric; Evanno, Gwénaëlle; Mai, Hoa Le; Martinet, Bernard; Duvaux, Odile; Bach, Jean-Marie; Conchon, Sophie; Judor, Jean-Paul; Perota, Andrea; Lagutina, Irina; Duchi, Roberto; Lazzari, Giovanna; Le Berre, Ludmilla; Perreault, Hélène; Lheriteau, Elsa; Raoul, Hervé; Volchkov, Viktor; Galli, Cesare; Soulillou, Jean-Paul

2016-01-01

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1–3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1–3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1–3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection. PMID:27280712

Dicty_cDB: FC-BS09 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS09 (Link to dictyBase) - - - Contig-U16215-1 FC-BS09Z (Li...nk to Original site) - - FC-BS09Z 626 - - - - Show FC-BS09 Library FC (Link to library) Clone ID FC-BS09 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS09Q.Seq.d/ Representative seq. ID FC-BS...09Z (Link to Original site) Representative DNA sequence >FC-BS09 (FC-BS09Q) /CSM/FC/FC-BS/FC-BS09Q.Seq....ignments: (bits) Value SSF360 (SSF360Q) /CSM/SS/SSF3-C/SSF360Q.Seq.d/ 854 0.0 FC-BS09 (FC-BS09Q) /CSM/FC/FC-BS/FC-BS

Dicty_cDB: FC-AI13 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI13 (Link to dictyBase) - - - Contig-U16263-1 FC-AI13F (Li...nk to Original site) FC-AI13F 507 - - - - - - Show FC-AI13 Library FC (Link to library) Clone ID FC-AI13 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI13Q.Seq.d/ Representative seq. ID FC-AI...13F (Link to Original site) Representative DNA sequence >FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq....nificant alignments: (bits) Value FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq.d/ 963 0.0 VSA730 (VSA730Q)

Dicty_cDB: FC-AK01 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AK01 (Link to dictyBase) - - - Contig-U15038-1 FC-AK01E (Li...nk to Original site) - - - - - - FC-AK01E 996 Show FC-AK01 Library FC (Link to library) Clone ID FC-AK01 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15038-1 Original site URL http://dict...s clone omyk-evn... 172 6e-79 AF401557_1( AF401557 |pid:none) Ictalurus punctatus...reticulum 4.0 %: vacuolar >> prediction for FC-AK01 is mit 5' end seq. ID - 5' end seq. - Length of 5' end s

Dicty_cDB: FC-AI03 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI03 (Link to dictyBase) - - - Contig-U15833-1 FC-AI03P (Li...nk to Original site) FC-AI03F 690 FC-AI03Z 651 FC-AI03P 1341 - - Show FC-AI03 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI03Q.Seq.d/ Representative seq. ID FC-AI...03P (Link to Original site) Representative DNA sequence >FC-AI03 (FC-AI03Q) /CSM/FC/FC-AI/FC-AI...li*l*ivtlskv*qswyqvfcslmrftcwi*nv fht*ivhwsqhwhql*flqpivaiv*skapitifnlhmaslwif*ivl*sfvpfhiitmk sfkfspfvpqlki

Dicty_cDB: FC-BR23 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BR23 (Link to dictyBase) - - - Contig-U15008-1 FC-BR23Z (Li...nk to Original site) - - FC-BR23Z 641 - - - - Show FC-BR23 Library FC (Link to library) Clone ID FC-BR23 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BR/FC-BR23Q.Seq.d/ Representative seq. ID FC-BR2...3Z (Link to Original site) Representative DNA sequence >FC-BR23 (FC-BR23Q) /CSM/FC/FC-BR/FC-BR23Q.Seq....9 0.0 SLA211 (SLA211Q) /CSM/SL/SLA2-A/SLA211Q.Seq.d/ 1029 0.0 FC-BR23 (FC-BR23Q) /CSM/FC/FC-BR/FC-BR2

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

NARCIS (Netherlands)

Engelberts, Patrick J.; Voorhorst, Marleen; Schuurman, Janine; van Meerten, Tom; Bakker, Joost M.; Vink, Tom; Mackus, Wendy J. M.; Breij, Esther C. W.; Derer, Stefanie; Valerius, Thomas; van de Winkel, Jan G. J.; Parren, Paul W. H. I.; Beurskens, Frank J.

2016-01-01

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20(+) B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab ')2 fragments, as well as C1q-binding-deficient IgG mutants,

Dicty_cDB: FC-AC21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AC21 (Link to dictyBase) - - - Contig-U15104-1 FC-AC21E (Li...nk to Original site) - - - - - - FC-AC21E 527 Show FC-AC21 Library FC (Link to library) Clone ID FC-AC21 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15104-1 Original site URL http://dict...ce KDSLDVIIFPEMVKLVGLTPNTMEKVLTYFQDNDTIDLSTFPMEIQVEQLSGKYIFICTH KQKDQRCGYCGPILVDQLRDQIKERSLEKEIQVFGTSHVGGHKY... Frames) Frame A: KDSLDVIIFPEMVKLVGLTPNTMEKVLTYFQDNDTIDLSTFPMEIQVEQLSGKYIFICTH KQ

Dicty_cDB: FC-IC0102 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC-IC (Link to library) FC-IC0102 (Link to dictyBase) - - - Contig-U16527-1 FC-IC01...02F (Link to Original site) FC-IC0102F 434 - - - - - - Show FC-IC0102 Library FC-IC (Link to library) Clone ...ID FC-IC0102 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16527-1 Original site URL http://dict... (bits) Value N AB088483 |AB088483.1 Dictyostelium discoideum gene for gamete and mating-type specific prote...oducing significant alignments: (bits) Value AB088483_1( AB088483 |pid:none) Dictyostelium discoideum gmsA g

Dicty_cDB: FC-BS24 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS24 (Link to dictyBase) - - - Contig-U16209-1 FC-BS24Z (Li...nk to Original site) - - FC-BS24Z 721 - - - - Show FC-BS24 Library FC (Link to library) Clone ID FC-BS24 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16209-1 Original site URL http://dict...nce. 52 9e-12 4 BQ096846 |BQ096846.1 IfHdk00487 Ictalurus furcatus head kidney cDNA library Ict...N SA ;, mRNA sequence. 44 1e-10 4 BM438323 |BM438323.1 IpLvr01076 Liver cDNA library Ictalurus punctatus cDN

Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active Multiple Sclerosis

DEFF Research Database (Denmark)

Laska, Magdalena Janina; Brudek, Tomasz; Nissen, Kari Konstantin

2012-01-01

of a capsid (Gag) protein of HERV-H/F origin by flow cytometry in peripheral blood mononuclear cells (PBMCs) from healthy controls and from MS patients with nonactive or active disease. There was a significant increase in HERV-H/F Gag expression in CD4(+) (P ...-fold increase in extracellular HERV-Fc1 RNA titers in patients with active MS compared with healthy controls (P

Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

Directory of Open Access Journals (Sweden)

Brian J. Franz

2015-01-01

Full Text Available Fc gamma receptor IIB (FcγRIIB is the only Fc gamma receptor (FcγR which negatively regulates the immune response, when engaged by antigen- (Ag- antibody (Ab complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft, a Category A biothreat agent. We utilized inactivated Ft (iFt as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO or wildtype (WT mice were challenged with Ft-live vaccine strain (LVS. While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.

Histopathology of IgG4-Related Autoimmune Hepatitis and IgG4-Related Hepatopathy in IgG4-Related Disease.

Science.gov (United States)

Nakanuma, Yasuni; Ishizu, Yoji; Zen, Yoh; Harada, Kenichi; Umemura, Takeji

2016-08-01

Immunoglobulin G4-related disease (IgG4-RD) is a systemic disease involving many organs; it includes IgG4-related sclerosing cholangitis and inflammatory pseudotumor in the hepatobiliary system. Two types of hepatic parenchymal involvement have been reported in IgG4-RD: IgG4-related autoimmune hepatitis (AIH) and IgG4-hepatopathy. Moreover, only three cases of IgG4-related AIH have been reported. Immunoglobulin G4-related AIH is clinicopathologically similar to AIH, except for an elevated serum IgG4 level and heavy infiltration of IgG4-positive plasma cells in the liver tissue. Interestingly, IgG4-related AIH can be complicated by well-known IgG4-RD(s). Immunoglobulin G4-hepatopathy, which includes various histopathological lesions encountered in the liver of patients with type I autoimmune pancreatitis, is classified into five histological categories: portal inflammation, large bile duct damage, portal sclerosis, lobular hepatitis, and cholestasis. Immunoglobulin G4-hepatopathy is currently a collective term covering hepatic lesions primarily or secondarily related to IgG4-related sclerosing cholangitis and type 1 autoimmune pancreatitis. In conclusion, the liver is not immune to IgG4-RD, and at least two types of hepatic involvement in IgG4-RD have been reported: IgG4-related AIH and IgG4-hepatopathy. Additional studies are required to clarify their precise clinical significance with respect to IgG4-RD and inherent liver diseases. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

International Nuclear Information System (INIS)

Ryu, T.; Davis, J.M.; Schwartz, K.A.

1990-01-01

The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

Science.gov (United States)

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

The Fc-receptor III of cultured human monocytes. Structural similarity with FcRIII of natural killer cells and role in the extracellular lysis of sensitized erythrocytes

NARCIS (Netherlands)