RESUMEN
Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.
Asunto(s)
Productos Biológicos/efectos adversos , Productos Biológicos/inmunología , Evaluación de Medicamentos/tendencias , Hipersensibilidad a las Drogas/diagnóstico , Proteínas/efectos adversos , Proteínas/inmunología , Algoritmos , Animales , Formación de Anticuerpos/fisiología , Congresos como Asunto , Evaluación de Medicamentos/legislación & jurisprudencia , Evaluación de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Guías como Asunto , Humanos , Inmunidad Innata/efectos de los fármacos , Legislación de Medicamentos , Modelos Biológicos , Procesamiento Proteico-PostraduccionalRESUMEN
Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/sangre , Inmunoglobulina G/química , Oligosacáridos/análisis , Vasculitis/inmunología , Estudios de Casos y Controles , Galactosa , Glicosilación , Humanos , Fragmentos Fab de Inmunoglobulinas/sangre , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/sangre , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/sangre , Procesamiento Proteico-PostraduccionalRESUMEN
A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Granulomatosis con Poliangitis/inmunología , Inmunoglobulina G/inmunología , Anciano , Afinidad de Anticuerpos/inmunología , Autoantígenos/inmunología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Humanos , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Mieloblastina , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Peroxidasa/inmunología , Receptores de IgG/análisis , Receptores de IgG/inmunología , Serina Endopeptidasas/inmunologíaRESUMEN
Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class. The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/inmunología , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Isotipos de Inmunoglobulinas/química , Oligosacáridos/metabolismo , Cristalización , Glicoproteínas/metabolismo , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/metabolismo , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Oligosacáridos/inmunología , Unión Proteica , Conformación Proteica , Difracción de Rayos XRESUMEN
The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/sangre , Autoanticuerpos/sangre , Enfermedades Autoinmunes/sangre , Galactosa/sangre , Inmunoglobulina G/química , Procesamiento Proteico-Postraduccional , Vasculitis/inmunología , Adolescente , Anciano , Anciano de 80 o más Años , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Especificidad de Anticuerpos , Presentación de Antígeno , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Síndrome de Churg-Strauss/sangre , Síndrome de Churg-Strauss/inmunología , Femenino , Glicosilación , Granulomatosis con Poliangitis/sangre , Granulomatosis con Poliangitis/inmunología , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Mieloblastina , Neutrófilos/enzimología , Neutrófilos/inmunología , Peroxidasa/inmunología , Polisacáridos/sangre , Serina Endopeptidasas/inmunología , Vasculitis/sangreRESUMEN
Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.
Asunto(s)
Antígenos CD/metabolismo , Inmunoglobulina G/metabolismo , Oligosacáridos/metabolismo , Receptores de IgG/metabolismo , Rastreo Diferencial de Calorimetría , Inmunoglobulina G/química , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) was expressed in a CHO-K1 parental cell line. The resulting IgG-Fc-linked carbohydrate was significantly alpha2,3-sialylated (53% of glycans), as indicated by normal- and reverse-phase HPLC analyses. Following transfection of a rat alpha2,6-sialyltransferase gene into this parental cell line, IgG-Fc-linked glycans were sialylated (60% of glycans) such that the ratio of alpha2,6- to alpha2,3-linked sialic acid was 0.9:1.0. By comparison, the wild-type IgG3 (F243) is minimally sialylated (2-3% alpha2,3-linked), thus suggesting that sialylation is controlled primarily by the protein structure local to the carbohydrate and that the two sialyltransferases compete to sialylate the nascent oligosaccharide. The additional alpha2,6-sialylation affected the function of the recombinant antibody. FA243 IgG3 having both alpha2,6 and alpha2,3-sialylation restored recognition to wild-type IgG3 levels for human FcgammaRI, FcgammaRII, and target cell lysis by complement. We discuss how sialylation linkage could modulate IgG function.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Sialiltransferasas/genética , Animales , Células CHO , Carbohidratos/análisis , Cromatografía Líquida de Alta Presión , Activación de Complemento , Cricetinae , Glicosilación , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Células K562 , Mutación , Ácido N-Acetilneuramínico/análisis , Nitrohidroxiyodofenilacetato/inmunología , Oligosacáridos/análisis , Ratas , Superóxidos/metabolismo , Transfección , Células U937 , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
The influence of sodium butyrate on the production and glycosylation of recombinant mouse/human chimeric antibody by transfected CHO-K1 cells was investigated. We selected cells expressing 'wild-type' antibody with a human IgG3 heavy chain and a mutant of this molecule in which Phe 243 is replaced by Ala. These proteins have previously been shown to exhibit very different glycoform profiles with the mutant IgG being comprised of glycoforms having a high galactose and sialic acid content. Cell culture with 0-5 mM butyrate was shown to effect a 2-4-fold increase in antibody production whilst the induction of apoptosis was observed in a dose-dependent manner. The optimal butyrate concentration was observed to be 2 mM. The glycoform profile of each antibody produced in the presence of butyrate was analyzed by HPAEC-PAD and shown to be unchanged, relative to that produced in the absence of butyrate. Biological activity was evaluated by the ability of the antibodies to trigger superoxide generation, through Fc gamma RI, and shown to be independent of production in the presence or absence of butyrate. A similar increase in production was observed for a high antibody-producing cell line when expanded in a hollow fibre bioreactor under low-serum conditions (1%). These results demonstrated that butyrate is of value for increasing the productivity of CHO-K1 for recombinant IgG and does not compromise either glycosylation or biological activity.
Asunto(s)
Butiratos/farmacología , Inmunoglobulina G/biosíntesis , Animales , Células CHO , Cricetinae , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Ratones , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , TransfecciónRESUMEN
The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (alpha-MHC Class II) lacking a CH3 domain pair (DeltaCH3-IgG1), single-chain Fv fusion proteins with Fc or a hinge-CH2 domain, Fc with/out a hinge, and a single CH2 domain. Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcgamma receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30-83%) or sialylated (9-21%) oligosaccharide chains with the highest levels observed for the single CH2 domain. These data show which architectural components influence IgG glycosylation processing and that the (CH3)2 pair is particularly influential. When MHC Class II bearing (JY) cells were sensitized with L243 DeltaCH3-IgG1, scFvFc, or scFvhCH2 they elicited superoxide production, from U937 cells, at levels of 35-45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhCH2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhCH2 homo-dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcgammaRI.
Asunto(s)
Inmunoglobulina G/genética , Oligosacáridos/biosíntesis , Superóxidos/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Secuencia de Carbohidratos , Vectores Genéticos , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Oligosacáridos/químicaRESUMEN
Neutrophils constitutively express FcgammaRIIa and FcgammaRIIIb receptors. Both receptors exhibit allelic variants which have different quantitative functional capacities: the biallelic FcgammaRIIa-R131 and -H131 alleles, and the neutrophil antigen (NA) NA1/NA2 alleles. ANCA activation of neutrophils requires ligation of FcgammaRIIa receptor, but recent data have shown that ANCA can also bind FcgammaRIIIb receptor. The aim of this study was to determine whether the FcgammaRIIIb polymorphism was a risk factor for the development of ANCA-associated systemic vasculitis, or the associated nephritis. FcgammaRIIIb receptor genotyping was determined by allele-specific polymerase chain reaction. Genomic DNA was extracted from 101 Caucasian patients with ANCA+ vasculitis (of whom 84 had renal disease) and 100 ethnically matched controls. Of the patients with ANCA+ systemic vasculitis, 71 had ANCA with specificity for proteinase 3 and 30 with specificity for myeloperoxidase (MPO). Overall no significant difference in genotype distribution or allele frequencies was found between patients and controls, or between patients with renal disease and controls. However, there was a trend for an increase in homozygosity for the NA1 allele in patients with a vasculitis and this was significant in patients who had anti-MPO antibodies. The FcgammaRIIIb receptor polymorphism is not a major factor predisposing to the development of ANCA+ systemic vasculitis or the associated nephritis. The over-representation of the FcgammaRIIIb homozygous NA1 allele in patients with anti-MPO antibodies may have implications for disease susceptibility.
Asunto(s)
Alelos , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Receptores de IgG/genética , Vasculitis/genética , Vasculitis/inmunología , Autoanticuerpos/inmunología , Humanos , Neutrófilos/inmunología , Polimorfismo GenéticoRESUMEN
Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.
Asunto(s)
Glicoproteínas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Oligosacáridos/inmunología , Receptores de IgG/inmunología , Rastreo Diferencial de Calorimetría , Glicosilación , Humanos , Desnaturalización Proteica , Transducción de Señal , Superóxidos/metabolismo , Temperatura , Células U937RESUMEN
Glycosylation of the Fc region of IgG (IgG-Fc) is essential for the full expression of Fc effector functions. The profound differences in functional activity observed between glycosylated and aglycosylated IgG have not previously been paralleled by the demonstration of large-scale structural changes. In the present study differential scanning microcalorimetry (DSMC) was used to investigate IgG-Fc glycoprotein stability and to determine the thermodynamic parameters for thermal unfolding, which will include a contribution from the intra-molecular oligosaccharide-protein interactions. The thermogram obtained for glycosylated IgG1-Fc yielded two clearly defined transitions whilst the glycosylated IgG4-Fc exhibited a single transition. The methodology was also able to reveal measurable differences in the stability of IgG4-Fc glycoforms differing by the presence or absence of terminal galactose residues; deglycosylated IgG4-Fc exhibited two transitions with evidence for destabilisation of the C(H)2 domain.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Rastreo Diferencial de Calorimetría , Glicosilación , Calor , Humanos , Conformación Proteica , Desnaturalización ProteicaRESUMEN
ANCA, implicated as having a pathogenic role in systemic vasculitis, can activate tumour necrosis factor-alpha (TNF-alpha)-primed neutrophils by cross-linking surface-expressed ANCA antigens with neutrophil FcgammaRIIa receptors to release reactive oxygen species. The FcgammaRIIa receptor exists as polymorphic variants, R131 and H131, which differ in their ability to ligate human IgG2 and IgG3. Neutrophils homozygous for the FcgammaRIIa-H131 allotype bind more efficiently to IgG3 than the FcgammaRIIa-R131 allotype and are the only human FcgammaR which bind IgG2. Our aim was to determine whether the homozygous FcgammaRIIa-H131 individuals are more susceptible to developing ANCA-associated systemic vasculitis and nephritis due to differential IgG binding and activation. FcgammaRIIa allotype was determined by both allele-specific polymerase chain reaction (PCR) and Southern blotting with allele-specific oligonucleotide probes end-labelled with 32P-gammaATP, after PCR amplification of genomic FcgammaRIIa DNA in 107 Caucasian patients with ANCA+ vasculitis (of whom 89 had renal disease) and 100 ethnically matched controls. Phenotyping of neutrophil FcgammaRIIa alleles was confirmed in some patients by quantitative flow cytometry using murine MoAbs 41H16 and IV.3. Of the patients with ANCA+ systemic vasculitis, 75 had ANCA with specificity for proteinase 3 and 32 with specificity for myeloperoxidase. Overall, no skewing in FcgammaRIIa allotypes was seen in patients compared with controls. No significant increase of the FcgammaRIIa-H131 allotype was found amongst patients irrespective of ANCA specificity, and no association between the FcgammaRIIa allotype and nephritis was found. Our data suggest that the FcgammaRIIa receptor allotype is not a major factor predisposing to the development of ANCA+ systemic vasculitis, or to nephritis.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Enfermedades Autoinmunes/inmunología , Isoformas de Proteínas/genética , Receptores de IgG/genética , Vasculitis/inmunología , Alelos , Animales , Anticuerpos Monoclonales/inmunología , Enfermedades Autoinmunes/genética , Southern Blotting , Síndrome de Churg-Strauss/genética , Síndrome de Churg-Strauss/inmunología , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Granulomatosis con Poliangitis/genética , Granulomatosis con Poliangitis/inmunología , Humanos , Inmunoglobulina G/inmunología , Ratones , Neutrófilos/inmunología , Fenotipo , Poliarteritis Nudosa/genética , Poliarteritis Nudosa/inmunología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Isoformas de Proteínas/inmunología , Especies Reactivas de Oxígeno , Receptores de IgG/inmunología , Estallido Respiratorio , Vasculitis/genéticaRESUMEN
Mutational analysis was used to determine the structural basis for the binding of the murine anti-idiotypic (anti-Id) monoclonal antibodies (MoAb) G6 and G8 to VH1-encoded antibodies. The MoAb G6 binds a cross-reactive idiotope present on the heavy (H) chains of immunoglobulins (Ig) encoded by 51p1-related gene segments, but not those encoded by hv1263-related gene segments. Gene segments 51p1 (DP-10) and hv1263 differ by only four amino acids; three in complementarity-determining region 2 (CDR2), and one in framework region 3 (FR3). The MoAb G8 also binds 51p1-related sequences, although it is undetermined whether G8 can bind hv1263-related sequences. In order to localise the Ids recognized by MoAbs G6 and G8 on 51p1-encoded antibodies, recombinant antibodies containing H-chain mutants were expressed in insect cells. Idiotypic analysis on the expressed recombinant proteins definitively localised the reactivity in each molecule. These studies should be important in the structural appreciation for critical serological reagents.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Monoclonales/genética , Sitios de Unión de Anticuerpos , Línea Celular , Mapeo Cromosómico , Humanos , Ratones , Datos de Secuencia Molecular , Spodoptera/citologíaRESUMEN
Human immunodeficiency virus (HIV)-infected persons manifest decreased antibody responses to pneumococcal polysaccharide vaccines. Since human antibody responses to polysaccharides are often restricted, the molecular structure of antibodies elicited by a 23-valent pneumococcal vaccine was analyzed. Anti-idiotypic reagents were used to detect V(H)1, V(H)3, and V(H)4 gene usage by antibodies to pneumococcal capsular polysaccharides in HIV-uninfected and HIV-infected subjects by ELISA. HIV-uninfected persons generated beta-mercaptoethanol-sensitive and -resistant antibodies to pneumococcal capsular polysaccharides expressing V(H)3 determinants recognized by the D12, 16.84, and B6 monoclonal antibodies; antibodies expressing V(H)1 determinants were not detected, and V(H)4 determinants were expressed by beta-mercaptoethanol-sensitive antibodies only; and HIV-infected subjects had significantly lower capsular polysaccharide-specific and V(H)3-positive antibody responses. These findings confirm decreased antibody responses to pneumococcal vaccination in HIV-infected persons and suggest that their poor responses may result from HIV-associated depletion of restricted B cell subsets.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antibacterianos/genética , Vacunas Bacterianas/inmunología , Infecciones por VIH/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Streptococcus pneumoniae/inmunología , Adulto , Animales , Anticuerpos Antibacterianos/inmunología , Sitios de Unión de Anticuerpos , Infecciones por VIH/sangre , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Mercaptoetanol , Ratones , Persona de Mediana Edad , Vacunas NeumococicasRESUMEN
Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolialectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.
Asunto(s)
Artritis Reumatoide/sangre , Inmunoglobulina G/sangre , Oligosacáridos/análisis , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Humanos , Inmunoglobulina G/aislamiento & purificación , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/fisiología , Inmunoglobulina G/química , Inmunoglobulina G/fisiología , Conformación Proteica , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Proteínas Portadoras/metabolismo , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Ligandos , Lectinas de Unión a Manosa , Oligosacáridos/química , Oligosacáridos/metabolismo , Receptores de IgG/metabolismo , Factor Reumatoide/metabolismoRESUMEN
Preferential expression of VH gene segments is evident within the adult human primary B-cell repertoire. The repertoire may be influenced by genetic factors, e.g. VH gene segment polymorphisms, or in a temporal manner due to the exposure to environmental antigens. The molecular characteristics of 15 autoreactive human monoclonal antibodies (MoAbs) are reported. All antibodies were of the IgM isotype, and 12 of the 15 were polyreactive and included rheumatoid factor type specificity, i.e. reactivity with IgG. Nine of the 15 MoAbs are products of VH3 gene segments, as evidenced by staphylococcal protein A binding; four of these express the cross-reactive idiotype recognized by the mouse MoAb 3H7 and are thus products of the VH26 gene segment. One of the five remaining VH3 gene products expresses the cross-reactive idiotypes recognized by the mouse MoAbs B6 and D12. V-gene family usage, determined by polymerase chain reaction (PCR) amplification of cDNA and further hybridization with family-specific oligonucleotide probes, confirmed the cross-reactive idiotype studies and showed that only VH3-gene-encoded proteins bound staphylococcal protein A. Five of the six non-VH3 gene segment products express the cross-reactive idiotype recognized by the mouse MoAb LC1 and could be assumed to be products of the VH4.21 gene segment; however, one human MoAb is shown to be the product of a VH2 gene segment. This is interesting because it turns LC1 from being an anti-cross-reactive idiotype antibody into an anticlan reagent.