Binding and activation of human precursor C1 by soluble aggregates of human and rabbit IgG

The capacities of soluble human and rabbit IgG aggregates to bind and to activate human C1 were compared. Aggregates prepared by incubation of purified IgG at 63 degrees C were fractionated by gel filtration and hemolytic assays were used to measure the binding and activation of isolated human precursor C1. The C1 binding and activation capacities of both human and rabbit IgG aggregates were highly dependent on their size. Human IgG aggregates had a slightly higher binding avidity for human C1 than rabbit IgG aggregates of comparable size, but no clear differences were found between their capacities to activate C1. Experiments with nonaggregated IgG also indicated that although human IgG binds human C1 somewhat more avidly, human and rabbit IgG do not differ in their capacities to initiate fluid-phase activation of the human classical complement pathway.     

Canine mast cell activation via human IgG1 and IgG4

BACKGROUND: We have reported that canine mastocytoma-derived CM-MC cells are activated via canine IgG and express a high-affinity IgG receptor (canine FcgammaRI). The predicted amino acid sequence of the canine FcgammaRI alpha subunit was found to be 72% similar to that of humans. These results suggest that canine FcgammaRI have binding activity with human IgG and led us to investigate CM-MC activation via canine FcgammaRI and human IgG. METHODS: The binding of human IgG to canine FcgammaRI was examined by flow cytometry using FITC-conjugated human IgG. [Ca2+]i increase or histamine release via canine FcgammaRI and the four human IgG subclasses was measured following aggregation of IgG-bound FcgammaRIs by anti-human IgG. To determine the binding activity of canine FcgammaRI with human IgG1 or IgG3, the displacement of 125I-labeled canine IgG from canine FcgammaRI was examined by unlabeled human IgG1 or IgG3. RESULTS: The fluorescence intensity of CM-MC cells was markedly (about 50 times) elevated by incubation with FITC-human IgG compared with the fluorescence of the control cells. A significant (p < 0.01) calcium response and histamine release were observed following aggregation of canine FcgammaRIs bound with human IgG1 or IgG4. 125I-labeled canine IgG was displaced from canine FcgammaRI by preincubation with unlabeled total human IgG or human IgG1 dose-dependently, whereas no displacement was detected by preincubation with human IgG3. CONCLUSIONS: Canine FcgammaRI possesses a significant binding activity with human IgG1 or IgG4, while IgG2 or IgG3 did not significantly react with canine FcgammaRI on CM-MC cells.     

A fully human antibody neutralising biologically active human TGFbeta2 for use in therapy.

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.     

Immunological studies of human placentae: subclass and fragment specificity of binding of aggregated IgG by placental endothelial cells.

Fluorescein-conjugated heat-aggregated human IgG binds to endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae. No binding was observed using native human IgG of heat-aggregated human albumin, IgM or IgA2. No inhibition of binding of heat-aggregated human IgG was observed by pre-treatment of placental tissue sections with native IgG or non-aggregated Fc fragments. The binding was blocked using heat-aggregated Fc fragments prepared from IgG1, IgG2, IgG3 and IgG4 myeloma proteins, but not with heat-aggregated human light chains, Fab and F(ab)2 fragments of human IgG, or with heat-aggregated human IgM and IgA2. It is suggested that the placental endothelial cell receptor for aggregated IgG may function to keep immune complexes from entering the foetal circulation.     

Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies

We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcgammaRI binding and monocyte activation [Eur. J. Immunol. 29 (1999) 2613]. Here, we detail interactions with FcgammaRIIa of the 131R and 131H allotypes and FcgammaRIIb. Lower hinge mutations caused large reductions in binding whereas modification of residues 327, 330 and 331 had less dramatic effects. However, like the wildtype IgG subclass binding hierarchies, the effect of the mutations varied between different receptors. We identified IgG1 variants which react with the activating receptor, FcgammaRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Manipulation of interactions with FcgammaRIIb separately from those with activating receptors provides potential for designing antibodies with novel and effective combinations of attributes. In addition, insight is gained into the evolution of functional differences in human IgG subclasses.     

Molecular recognition of human insulin receptor by autoantibodies in a human serum

Immune IgG was obtained from a patient's serum with autoantibodies against human insulin receptor (HIR). The binding activity of these autoantibodies to seven synthetic peptides of the alpha-subunit of HIR was examined by radioimmuno-adsorbent titrations. Autoantibodies were bound by two of these peptides, namely alpha 277-299 and alpha 705-731, and this binding was not inhibited by a very large (1,000 x) molar excess of normal human IgG. Furthermore, none of the peptides exhibited any binding activity towards 125I-labelled normal human IgG. Therefore, it was concluded that the regions alpha 277-299 and alpha 705-731 contain autoantigenic sites of HIR. In addition, region alpha 655-670 might constitute a minor antigenic site of HIR. Localization of these autoantigenic sites will permit the molecular investigation of the insulin-like activity of anti-HIR autoantibodies.     

The spontaneous ability of normal human IgG to inhibit the Fc receptors of normal human monocytes is related to their binding capacity to lectins

The lectin-binding capacity of 96 normal human IgG, assessed by solid-phase radioimmunoassay, strikingly varied according to the lectin considered. Indeed, half of the IgG samples exhibited peanut agglutinin (PNA)- and pokeweed mitogen-specific binding capacities superior or equal to 4%, whereas less than 15% of IgG similarly bound to concanavalin A (Con A) and to phytohemagglutinin. The ability of those IgG to inhibit the Fc receptor (Fc-R) function of human monocytes, measured by a classical rosette assay, was inversely correlated to their binding ratios to PNA and Con A only. By affinity chromatography, three groups of IgG were separated: the IgG purified on agarose-PNA columns slightly reduced the Fc-R function (40-45% inhibition); the IgG purified on Sepharose-Con A columns exhibited the highest inhibitory properties (80-85% inhibition); the IgG that did not bind to PNA and Con A columns possessed intermediate inhibitory properties (65-70% inhibition). The different effect of IgG on Fc receptors was conserved when monocytes were first treated by trypsin and was unrelated to their specific binding to human monocytes, to their subclasses, and to their C1q- or protein A-binding capacities. Incubation of monocytes with D-galactose (10 mM) significantly improved their capacity to form IgG rosettes, whereas their incubation with D-mannose (10 mM) significantly reduced the Fc-R function. Scatchard plots of 125I-IgG1 myeloma protein binding to monocytes were linear under basal conditions, as well as after a prior incubation of the cells with D-galactose or D-mannose. Monocytes bound about 16,000 molecules of IgG1 per cell in each instance. In contrast, the mean association constant (Ka) for IgG1 binding was 2.59 +/- 0.50 X 10(8) M-1 under basal conditions, 4.4 +/- 0.75 X 10(8) M-1 after D-galactose incubation, and 1.35 +/- 0.50 X 10(8) M-1 after D-mannose incubation. These data suggest that the level of human monocyte Fc-R function blockade induced by human IgG depends mainly on the presence of "accessible" galactosyl or mannosyl residues in the Fc domain and that the modulation of the Fc-R function induced by these carbohydrates is due to a change in the affinity rather than in the number of single class of high-affinity binding sites.     

The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages

We examined the binding characteristics of human monocytes and macrophages with the IgG ligands, human monomeric IgG and a small human IgG aggregate, trimeric IgG. Our purpose was to utilize fresh monocytes, in vitro cultured monocytes, and alveolar macrophages in direct and indirect binding experiments. Freshly isolated monocytes expressed only a single binding site for IgG monomer and IgG trimer. In contrast, in vitro cultured monocytes, gamma-interferon-treated monocytes, and freshly isolated alveolar macrophages expressed a single binding site for IgG monomer and, in addition, a high and low affinity binding site for IgG trimer. The high affinity binding site for IgG trimer (Kd approximately equal to 1 nM) appeared identical to the binding site for IgG monomer. The low affinity binding site for IgG trimer (Kd = 50 to 250 nM) appeared to be due to Fc gamma RII, because antibody to Fc gamma RII inhibited its expression. Since Fc gamma RII, in contrast to Fc gamma RI, does not bind monomeric IgG, the data suggest that this low affinity receptor for trimeric IgG, Fc gamma RII, can bind low molecular weight circulating immune complexes at concentrations 10- to 100-fold lower than Fc gamma RI. Thus, these studies suggest that at 37 degrees C, macrophage Fc gamma RII may play a functional role in the recognition of small molecular weight immune complexes.     

The double reactivity of a human monoclonal rheumatoid factor to IgG and histones is related to distinct binding sites

The structural basis of the double reactivity of a human monoclonal rheumatoid factor (RF) with both human IgG and histones H1 and H3 was investigated by means of competitive inhibition experiments. The monoclonal RF binding to solid-phase histones was inhibited by increasing concentrations of heat-aggregated IgG. However, increasing concentrations of purified histones were almost unable to reduce the RF binding to solid-phase IgG. Inhibition of antigen binding with two murine monoclonal anti-idiotopes reacting with distinct idiotopes on the monoclonal RF indicated that the fixation to the different antigens was mediated by distinct binding sites. This result was confirmed by showing the selective sensitivity to mild acid treatment of the histone binding site but not of the IgG binding site. This report provides a structural basis for the existence of polyfunctional combining regions on a human autoantibody.     

The effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes

We report the effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes. Cytochalasin B inhibits EA- but not EAC-rosette formation, and neither colchicine nor vinblastine have a measurable effect on either reaction. Also, cytochalasin B inhibits the binding of heat-aggregated IgG by human lymphocytes. The inhibition caused by cytochalasin B is reversible, and the cells recover to normal values within 30 min.     

Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52.

Activation of the complement cascade by immunoglobulin G (IgG) plays a major role in the host defense against pathogens. Using recombinant human antibodies specific for the leucocyte antigen CD52, different allotypes of human IgG1 subclass were compared for their ability to activate human complement. In addition the roles of the different length hinge regions of IgG1 and IgG3 were investigated. It was found that the naturally occurring allotypes G1m(a,z) and G1m(f), and one artificially created isoallotype, G1m(null), did not significantly differ in their overall ability to cause cell lysis. However, some differences in binding of individual components of the classical activation pathway were detected. More of the complement component C1s seemed to be associated with the allotype G1m(f), although this did not result in an overall improvement in lytic potency. In this system the wild-type IgG3 was found to be less effective in complement lysis than IgG1. By shortening the hinge region of IgG3 to resemble that of an IgG1 antibody, increased complement binding was observed compared with that of wild-type IgG3 and the IgG1 allotypes. The overall lytic potency of the antibody was also improved compared with wild type IgG3 and it was also slightly more effective than the IgG1 allotypes.     

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2.     

Localisation of the monocyte-binding region on human immunoglobulin G

Earlier studies, which provided indirect evidence for the involvement of the C gamma 2 domain of human immunoglobulin G (IgG) in human immunoglobulin G (IgG) in human monocyte binding, have been extended to further localise the site of interaction on human IgG. A number of IgGs from several different species and fragments of human IgGs were assayed for ability to inhibit the interaction of radio-labelled human IgG and the human monocyte. By comparison of the amino-acid sequences of those IgGs found to exhibit relatively tight, intermediate or weak binding to human monocyte Fc receptors we are able to postulate a possible monocyte-binding site on human IgG. In addition, the results have implications for the applicability of monoclonal antibodies and antisera when used in the presence of human monocytes and possibly macrophages.     

A mouse monoclonal antibody that reacts specifically with immune-complexed human IgG

After human IgG binds to antigen, it attains biological functions that are not properties of monomeric, uncomplexed IgG, including the ability to activate complement and to bind to cellular receptors. Associated with antigen binding, we have recently demonstrated that IgG itself has neoantigenic epitopes. Antibodies to these neoantigens on immune-complexed IgG may represent a significant proportion of circulating anti-human IgG in rabbits immunized with immune complexes. In contrast, mice immunized in an identical fashion have very little circulating anti-neoantigen antibody. This is true whether the mice are genetically easy to tolerize to monomeric human IgG (DBA/2 and C57Bl/6) or difficult to tolerize (BALB/c). Fusions were made between the NS-1 myeloma cell line and spleen cells from mice of each strain, which had been made tolerant to monomeric human IgG and then immunized with immune complexes containing IgG. Like the serum antibody, antibodies made by these fusions showed little specificity for immune complexes since 99% of the hybridoma antibodies that recognized IgG in immune complexes also bound to uncomplexed IgG. Only 1 hybridoma produced antibody that preferentially recognized human IgG in immune complexes. This antibody, called CE9, is an IgM that binds to IgG in plate-bound immune complexes with 100-1000-fold greater avidity than it does to plate-bound uncomplexed IgG. Because CE9 will not bind to immune complexes made with F(ab')2 antibody, the epitope it recognizes requires the Fc fragment of IgG. The minimal binding of CE9 to uncomplexed IgG is easily inhibited by soluble aggregates of IgG, but binding to immune complexes is not inhibited by aggregated IgG. CE9 does recognize fluid-phase immune complexes as well as solid-phase immune complexes. We conclude that, while mice produce much less anti-immune complex antibody than rabbits, anti-neoantigen is still a component of their response to immunization with immune complexes. Using hybridoma techniques to amplify these anti-neoantigen antibodies, we have shown that they resemble rheumatoid factors in their isotype and binding properties.     

Autoantigen Ro52 directly interacts with human IgG heavy chain in vivo in mammalian cells

Previously, when we used in vivo yeast two-hybrid and in vitro protein-protein interaction analyses, we demonstrated a direct interaction between autoantigen Ro52 and the human IgG heavy chain. This interaction occurred in the absence of antibody-antigen specific interaction. Here, by employing a novel strategy, we further demonstrated that Ro52 co-localized with IgG in transfected mammalian cells. The co-localization was specific to IgG1 but not IgG3. Co-immunoprecipitating IgG with Ro52 from transfected cell lysates suggested that protein complex containing Ro52 and IgG contributed to the in vivo co-localization. In addition, IgG from normal human serum was shown to bind to the surface of apoptotic keratinocytes and the binding could be competitively blocked by 50-fold excesses of IgG1, not IgG3. With a direct binding study, we also demonstrated that IgG1 could bind to the surface of apoptotic cells while IgG3 bound barely. This binding was not competed by Fcgamma fragments indicating a non-Fcgamma receptor mediated interaction. Finally, in a competition analysis the addition of GST-RFP could reduce the IgG binding to the cell surface. Thus, we suggested that the binding of IgG to the apoptotic keratinocytes might be mediated through the interactions with the surface exposed Ro52. The potential role of forming this protein complex on the apoptotic cells will be discussed.     

Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities.

Subclasses of human IgG have a range of activity levels with different effector systems but each triggers at least one mechanism of cell destruction. We are aiming to engineer non-destructive human IgG constant regions for therapeutic applications where depletion of cells bearing the target antigen is undesirable. The attributes required are a lack of killing via Fcgamma receptors (R) and complement but retention of neonatal FcR binding to maintain placental transport and the prolonged half-life of IgG. Eight variants of human IgG constant regions were made with anti-RhD and CD52 specificities. The mutations, in one or two key regions of the CH2 domain, were restricted to incorporation of motifs from other subclasses to minimize potential immunogenicity. IgG2 residues at positions 233 - 236, substituted into IgG1 and IgG4, reduced binding to FcgammaRI by 10(4)-fold and eliminated the human monocyte response to antibody-sensitized red blood cells, resulting in antibodies which blocked the functions of active antibodies. If glycine 236, which is deleted in IgG2, was restored to the IgG1 and IgG4 mutants, low levels of activity were observed. Introduction of the IgG4 residues at positions 327, 330 and 331 of IgG1 and IgG2 had no effect on FcgammaRI binding but caused a small decrease in monocyte triggering.     

FcRn mediates elongated serum half-life of human IgG in cattle

IgG has the longest survival time in the circulation of the Ig classes and the lowest fractional catabolic rate. The neonatal Fc receptor (FcRn) plays an important role in regulating these processes. Recently, we have cloned the bovine neonatal Fc receptor (bFcRn) alpha chain and detected its expression in various epithelial cells which are mediating IgG secretion. However, its function in IgG homeostasis has not been investigated. In the current study, we analyzed the binding affinity of bovine and human IgGs to bFcRn using surface plasmon resonance and by in vitro radioreceptor binding assays. As human IgG binds stronger to the bFcRn, than bovine IgG at pH 6, we subsequently analyzed its catabolism in normal and transchromosomic calves that produce human Igs. Pharmacokinetic studies showed that human IgG had approximately 33 days serum half-life both in normal and transchromosomic calves, which is more than two times longer than its bovine counterpart. We also demonstrate FcRn expression in endothelial cells and in the kidney which are supposed to be involved in IgG metabolism. These data suggest that bFcRn is involved in IgG homeostasis in cattle and furthermore, that the transchromosomic calves producing human Igs can effectively protect their human IgGs which have implications for successful large-scale production of therapeutic antibodies.     

Immunological studies of human placentae. Binding of complexed immunoglobulin by stromal endothelial cells.

Endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae bind fluorescein-conjugated heat-aggregated human IgG. Soluble immune complexes of sheep anti-human albumin also bind in a pattern that is similar to that of aggregated human IgG, but native human IgG is not bound by placental endothelial cells. Aggregated IgG binding, unlike soluble immune complexes, is blocked by pretreatment of the sections with antiserum to human IgM. Cross-blocking experiments with aggregated IgG and immune complexes suggest that they may be bound by different receptors.     

Bovine IgG1, but not IgG2, binds to human B cells and inhibits antibody secretion

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.