Identification of a unique IgG Fc binding site in human intestinal epithelium

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.     

Identification of multiple isoforms of the low-affinity human IgG Fc receptor

Two varieties of similar, but structurally distinct, cDNA clones for the human low-affinity receptors for the Fc portion of immunoglobulin G (FcγRII) have been isolated. One type of clone was obtained from human B lymphocytes, and the other from PHA-activated peripheral T cells and monocytes. Transfection of both prototype clones into Cos-7 cells and subsequent specific staining with monoclonal antibodies of the CDw32 group confirmed the identification of the gene products. The nucleotide sequence of the cDNA clone from B lymphocytes contains an open reading frame that encodes a protein of relative mass (M _r) 27000 with an extracellular domain of 179 amino acids containing three potential N-glycosylation sites, a 26 amino acid transmembrane domain, and a 44 amino acid cytoplasmic domain. The clones from peripheral T cells and monocytes both encoded a protein ofM _r 31000 with a 179 amino acid extracellular domain containing two potential N-glycosylation sites and a 26 amino acid transmembrane domain. The two types of clones had similar sequences in their immunoglobulin-like extracellular and transmembrane domains, but differed in their leader sequences and 3′-untranslated regions. The most notable difference between the clones was the presence of a distinctive 76 amino acid cytoplasmic domain in those isolated from T cells and monocytes.     

Identification of a lymphocyte-activating pentapeptide sequence in the Fc region of human IgG1

The synthetic peptide p23, representing residues 335 to 357 in the Fc region of human IgG1, was previously shown to induce Ig secretion in murine spleen cell cultures. In this report, overlapping peptides based on the sequence of p23 were synthesized to further map the active site in this molecule. The results from these studies indicate that leu-pro-pro-ser-arg (residues 351 to 355) retained the B cell differentiation-inducing properties of p23; however, expression of activity by this sequence was markedly influenced by N-flanking sequences. By using T cell-depleted spleen cell cultures, it was determined that at least two signals are required for p23-induced Ig secretion: one supplied by p23 directly and one supplied by a T cell-replacing factor present in p23-conditioned spleen cell supernatants. Both signals were mapped into the sequence leu-pro-pro-ser-arg. However, the latter signal, but not the former signal, again appeared to be influenced by sequences proximal to the active site. These data indicate that although the leu-pro-pro-ser-arg sequence is able to provide both required signals for p23-induced Ig secretion in spleen cell cultures, there may be subtle differences in how the cell types involved in this response interact with and/or are activated by this sequence.     

Identification of a receptor for IgG on human granulocytes

The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected between these carboxylating systems with respect to their affinity for vitamin K and warfarin. Most of the endogenous substrates had some affinity towards antiprothrombin or antifactor X.     

Subclass specificity of the Fc receptor for human IgG on K562

The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).     

Cloned and expressed human Fc receptor for IgG mediates anti-CD3-dependent lymphoproliferation

We have utilized gene transfer experiments to investigate the role of a human monocyte receptor for IgG (Fc gamma RII) in mouse IgG1 anti-CD3 (Leu 4)-induced lymphoproliferation in vitro. Mouse Ltk- cells expressing human Fc gamma RII or a mutant of Fc gamma RII lacking the entire cytoplasmic domain of the receptor mediate anti-CD3-induced lymphoproliferation in cultures of adherent cell-depleted human PBMC. Expression of an Fc gamma RII mutant lacking transmembrane and cytoplasmic domains (soluble Fc gamma RII) in COS7 cells yielded a secreted receptor which retained affinity for IgG, even in the absence of the mutant receptor's N-linked oligosaccharides. Soluble Fc gamma RII inhibits rosette formation by human IgG-sensitized RBC and the Fc gamma RII-bearing cell line K562, but does not sitmulate anti-CD3-induced lymphoproliferation under the conditions tested.     

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region

The IgG binding Fcgamma receptors (FcgammaRs) play a key role in defence against pathogens by linking humoral and cell-mediated immune responses. Impaired expression and/or function of FcgammaR may result in the development of pathological autoimmunity. Considering the functions of FcgammaRs, they are potential target molecules for drug design to aim at developing novel anti-inflammatory and immunomodulatory therapies. Previous data mostly obtained by X-ray analysis of ligand-receptor complexes indicate the profound role of the CH2 domain in binding to various FcgammaRs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity FcgammaRs, like FcgammaRIIb and FcgammaRIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231-298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble FcgammaRIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble FcgammaRIIb, as well as to FcgammaRs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg(255)-Ser(267) sequence of IgG1 is implicated in the binding to FcgammaRIIb. In addition we found that peptides corresponding to the Arg(255)-Ser(267), Lys(288)-Ser(298) or Pro(230)-Val(240) when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNFalpha, a pro-inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating FcgammaRs, and mediate FcgammaR dependent function. Peptide Arg(255)-Ser(267) can also be considered as a lead for further functional studies.     

A receptor for the Fc fragment of human IgG in the causative agent of tularemia

In 4 Francisella tularensis strains varying in virulence a receptor to Fc site of human IgG has been detected. This receptor consists of two active components with molecular weights of 67,000 and 40,000, competing for binding on Fc site of human IgG with Staphylococcus aureus protein A.     

Structural polymorphism of the human monocyte 40 kilodalton Fc receptor for IgG

The 40 kD monocyte Fc receptor for IgG is capable of binding murine IgG1 and of supporting an IgG1 anti-T3 T lymphocyte proliferative response among approximately 80% of Caucasian individuals (responders), whereas the 40 kD Fc receptor on monocytes of the remaining individuals (nonresponders) is incapable of interacting with murine IgG1. By using a monoclonal antibody (mab IV3) that reacts with the 40 kD receptor, we found that the monocyte 40 kD receptors from responder and nonresponder individuals cannot be distinguished by either electrophoretic mobility on SDS-polyacrylamide gels, or by the number of receptors per cell as determined by indirect immunofluorescence. However, isoelectric focussing of the purified radioiodinated 40 kD receptor revealed that the monocyte receptor from all of four nonresponder individuals evaluated has a single distinctive pattern of multiple, regularly spaced bands, whereas the pattern of the 40 kD monocyte receptor from 11 responder individuals is of two sorts. One (seen in four of 11 responders) consists of multiple, regularly spaced bands that are asynchronous with the nonresponder pattern, and the other (seen in seven of 11 responders) consists of multiple bands that correspond in mobility to all of the bands of both of the other two patterns. The incidence of these three patterns suggests that the 40 kD Fc receptor is encoded by a single structural gene with two alleles, both of which are expressed.     

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences

Many biological functions, including control of the homeostasis and maternofetal transfer of serum gamma-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t(1/2). To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with DeltaDeltaG (DeltaG(wild type) - DeltaG(mutant)) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.     

Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.     

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcγ receptors (FcγRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcγRI with very high affinity, but not to FcγRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcγRI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed FcγRI-positive macrophage-like U937 cells but not FcγRI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to FcγRIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc's unique FcγRs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcγRI and related diseases, including cancer.     

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcγ receptors (FcγRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcγRI with very high affinity, but not to FcγRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcγRI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed FcγRI-positive macrophage-like U937 cells but not FcγRI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to FcγRIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc''s unique FcγRs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcγRI and related diseases, including cancer.     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

T15 group A streptococcal Fc receptor binds to the same location on IgG as staphylococcal protein A and IgG rheumatoid factors

Previous work has shown that IgG rheumatoid factors (RF) bind to the C gamma 2-C gamma 3 interface region of human IgG in the same area that binds staphylococcal protein A (SPA). Group A, C, and G strains of Streptococci possess Fc receptors that bind to IgG but not to fragments containing only the C gamma 2 or C gamma 3 domains. This work describes the binding site location on human IgG for the binding of the isolated Fc receptor from the T15 strain of a Group A streptococcus and its relationship to the site that binds SPA and the IgG RF. The isolated T15 Fc receptor (T15) with a molecular mass of 29.5 kD inhibited the binding of IgG RF to IgG. The binding of T15 itself to IgG was strongly inhibited by SPA (42.0 kD) and its monovalent fragment D (7 kD). Human IgG fragments consisting of the C gamma 3 domains did not inhibit the binding of T15 to IgG, whereas those with both domains were effective inhibitors. T15 did not bind to rabbit IgG fragments consisting of either the C gamma 2 or C gamma 3 domains, but did bind to those with both domains. An IgG3 myeloma protein was a poor inhibitor and has been shown to bind poorly to the IgG RF. Most IgG3 myeloma proteins did not bind to SPA. The substitution of Arg and Phe for His 435 and Tyr 436 is responsible for the poor binding of IgG3 to SPA and to the IgG RF. Chemical modification of His or Tyr on IgG reduced its ability to inhibit the binding of T15 to IgG. Reversal of the chemical modifications with hydroxylamine resulted in near complete restoration of inhibitory capacity. This information, collectively, coupled with the known positions in space of the His and Tyr residues in the C gamma 2-C gamma 3 interface region, verified that both His 435 and Tyr 436, and possibly His 310 and 433, are involved. These residues are also involved in binding SPA and the IgG RF. These data therefore indicate that the T15 Group A Streptococcal Fc receptor binds to the same location on the Fc of IgG as SPA and the IgG RF. The biologic relevance of these similarities between bacterial cell wall Fc receptors and IgG RF are not yet apparent, but suggest that RF could bear the internal image of these bacterial structures.     

Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus

A receptor for the Fc region of immunoglobulin G was extracted from a group C streptococcus, purified and physicochemically characterized. The Fc receptor was extracted in high yield by lysis of the bacteria after infection with bacteriophage. The soluble receptor was purified to functional homogeneity by sequential chromatography on cellulose phosphate, DEAE, and selective elution from a column of immobilized human IgG. Four hundred micrograms of the functionally pure protein was obtained per gram (wet weight) of bacteria extracted. The affinity-purified receptor was functionally homogeneous in binding to the Fc region of human IgG; however, the product was heterogeneous on both non-denaturing and SDS polyacrylamide gels. Four major protein bands were observed, with the predominant form of the Fc receptor having an m.w. of 64,000 daltons. Antibody prepared against the major Fc receptor protein ( FcRc -II) was capable of reacting with all the fractions and completely inhibiting functional activity. The results of the competitive binding studies suggest that the purified Fc receptor behaves as a single receptor, and that the differences in charge and size were probably due to covalently bound cell wall constituents.     

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.     

A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.