Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants

The binding site specificity of 12 monoclonal and 11 polyclonal IgM rheumatoid factors (RF) isolated from human plasma or serum has been studied. All IgM RF bound best to sites on IgG and intact Fc. The monoclonal IgM RF did not bind at all to fragments lacking the C gamma 2 or C gamma 3 domains. In contrast, low level binding to the pFc' fragment, composed of the C gamma 3 domain, was seen with seven IgM RF, mainly from patients with rheumatoid arthritis (RA). IgG1 binding appeared to be a requisite specificity of all human IgM RF. IgM RF binding to IgG3 subclass was common among the monoclonal IgM RF. Most RA polyclonal IgM RF but only 2 of the monoclonal IgM RF possessed the IgG1, 2 and 4 binding pattern. Monoclonal IgM RF which bound best to histidine-modified IgG also bound well to IgG3. The 7-kDa fragment D of staphylococcal protein A inhibited the IgG binding of most monoclonal and to a lesser degree polyclonal IgM RF. Thus, the results indicate that the C gamma 2-C gamma 3 interface region of IgG contains the predominant determinants for monoclonal and polyclonal IgM RF. For some monoclonal IgM RF the binding site, even though at the interface of the C gamma 2 and C gamma 3 domains, is not the staphylococcal protein A site. Furthermore, polyclonal IgM RF possess specificities not encountered among the monoclonal IgM RF. These specificities may have special     

Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.     

Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Modulation of human rheumatoid factor-specific lymphocyte responses with a cross-reactive anti-idiotype bearing the internal image of antigen

Rabbit anti-idiotypic antibodies to human rheumatoid factor (RF) autoantibodies were isolated by affinity chromatography on rabbit anti-human IgG Fc Sepharose 4B. The anti-idiotypic antibodies bore the "internal image" of the antigen, human IgG. They reacted specifically with multiple human monoclonal and polyclonal IgM-RF, independent of any particular light or heavy chain amino acid sequence. The anti-idiotypes did not react with IgM or IgG proteins lacking RF activity. The present experiments determined the potential of the "internal image" antibodies to modulate in vitro lymphocyte functions. The addition of anti-idiotypic antibody to peripheral blood mononuclear cell cultures from patients with rheumatoid arthritis elicited lymphocyte proliferation, but not RF synthesis. The antibody did not induce the proliferation of lymphocytes from a normal individual. Moreover, the anti-idiotype specifically suppressed IgM-RF secretory responses when preincubated with B cells before co-culture with autologous pokeweed mitogen-activated T cells. The data show that the anti-idiotypic antibodies with the "internal image" of antigen are capable of interacting with B cell receptors in an antigen-restricted manner, and possess specific immunomodulatory properties.     

A monoclonal anti-idiotype specific for human polyclonal IgM rheumatoid factor.

One of the hallmarks of rheumatoid arthritis (RA) is the production of high titers of rheumatoid factor (RF) antibody directed against the Fc portion of IgG. Anti-Id that recognize the majority of monoclonal RF from patients with B cell dyscrasias are reactive with only 1 to 2% of these polyclonal RF from RA patients. We describe a new monoclonal anti-Id, 4C9, that recognizes a L chain determinant on polyclonal IgM RF from patients with RA but does not recognize a panel of monoclonal RF from patients with B cell malignancies. 4C9 reactivity is found in the serum of 34/43 RF-positive RA patients and in 12/12 RF-positive synovial fluids, but in only 1/14 RF-negative sera from RA patients and 1/22 sera containing monoclonal IgM RF. 4C9 reactivity is highly enriched in purified IgM RF from nine RA patients and represents a variable percentage of total IgM RF up to a maximum of 23%. Furthermore, 4C9 reactivity is enriched in the synovial fluid of three of five RA patients compared with serum, suggesting that 4C9-reactive IgM RF are synthesized within the joint. IgG RF from RA synovial fluids are not 4C9 reactive, indicating either that different genes are used to encode IgM and IgG RF in RA patients, or that IgG RF have somatically mutated away from idiotypic reactivity.     

Revisiting the role of glycosylation in the structure of human IgG fc

Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 ? resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the C(H)2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between C(H)2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (R(g)) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger R(g) than glycosylated Fc, suggesting a more open C(H)2 orientation under these conditions. Moreover, the R(g) of aglycosylated Fc was reduced by mutations at the C(H)2-C(H)3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.     

Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Mapping of four light chain-associated idiotopes of a human monoclonal rheumatoid factor

In an effort to analyze both IgM rheumatoid factor (RF) repertoire and regulation of RF production in humans, we developed a panel of four mouse monoclonal antibodies (mAb) defining distinct K light chain-associated idiotopes (id) of a human monoclonal IgM RF (Alt). These mAb (A75, AM1, AM2, AM3) had equivalent reactivities with the immunizing RF during classic inhibition of antigen-binding assays. These anti-id reagents were reacting to neither other tested monoclonal IgM RF nor normal polyclonal IgM. It was possible to distinguish the id defined by the mAb from the results of four sets of experiments: dissociation of Alt RF heavy (H) and light (L) chains showed that A75, AM1, and AM2 reacted to id located on the L chain, whereas AM3 defined a conformational RF id; recombination experiments of H and L chains showed that A75 and AM2 reacted well with both homologous (Alt H + Alt L) and heterologous (Alt L + unrelated H) recombinants, whereas AM1 reacted better with the homologous recombinant than with the heterologous one; the relative affinities of the mAb were drawn from their ability to shift already bound labeled Alt RF from solid phase IgG; and radiolabeling of two mAb (A75 and AM3) and experiments of inhibition of id binding with cold anti-id and cold anti-CK showed that A75 recognized a proximal id (close to the K constant region), whereas AM3 defined a more distal id, AM2 and AM1 being located between A75- and AM3-defined sites. This topographic mapping of K light chain-associated id of a human RF with anti-id of known relative affinities could help in studying idiotypic regulation in humans.     

Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue.

Using hybridoma technology we established a panel of human monoclonal rheumatoid factors (RF) from the synovial tissues of two patients with rheumatoid arthritis (RA), and one patient with polyarticular juvenile RA. Nucleotide sequence analysis of the V regions of these RF indicates that two independently derived antibodies from one of the RA patients are clonally related. One of these antibodies appears to be close to germ-line configuration, whereas the other has accumulated a total of 36 substitutions in both H and L chains. Measurements of the affinity for human IgG of the two RF show that the extensively mutated RF has 100-fold higher affinity for IgG than the RF close to germline. These findings indicate that IgM RF in RA can undergo affinity maturation and suggest that certain RF may be the product of an Ag-driven immune response.     

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Isolation and characterization of IgG1 with asymmetrical Fc glycosylation

N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemi-glycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease (～20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The decreased binding affinities to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.     

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).     

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.     

Thermodynamic Stability and Functional Activity of Tumor-Associated Antibodies

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2–CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.