Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.     

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C_H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.     

Monovalent IgG4 molecules

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

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody

Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.     

Structure of the Murine Unglycosylated IgG1 Fc Fragment

A prototypic IgG antibody can be divided into two major structural units: the antigen-binding fragment (Fab) and the Fc fragment that mediates effector functions. The IgG Fc fragment is a homodimer of the two C-terminal domains (C_H2 and C_H3) of the heavy chains. Characteristic of the Fc part is the presence of a sugar moiety at the inner face of the C_H2 domains. The structure of this complex branched oligosaccharide is generally resolved in crystal structures of Fc fragments due to numerous well-defined sugar-protein interactions and a small number of sugar-sugar interactions. This suggested that sugars play an important role in the structure of the Fc fragment. To address this question directly, we determined the crystal structure of the unglycosylated Fc fragment of the murine IgG1 MAK33. The structures of the C_H3 domains of the unglycosylated Fc fragment superimpose perfectly with the structure of the isolated MAK33 C_H3 domain. The unglycosylated C_H2 domains, in contrast, approach each other much more closely compared to known structures of partly deglycosylated Fc fragments with rigid-body motions between 10 and 14 Å, leading to a strongly “closed” conformation of the unglycosylated Fc fragment. The glycosylation sites in the C′E loop and the BC and FG loops are well defined in the unglycosylated C_H2 domain, however, with increased mobility and with a significant displacement of about 4.9 Å for the unglycosylated Asn residue compared to the glycosylated structure. Thus, glycosylation both stabilizes the C′E-loop conformation within the C_H2 domain and also helps to ensure an “open” conformation, as seen upon Fc receptor binding. These structural data provide a rationale for the observation that deglycosylation of antibodies often compromises their ability to bind and activate Fcγ receptors.     

Extending human IgG half-life using structure-guided design

Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.     

Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys

Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.     

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Engineering the interactions between a plant‐produced HIV antibody and human Fc receptors

Plants can provide a cost‐effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic α1,3‐fucose and β1,2‐xylose residues and glycans extended with terminal β1,4‐galactose. Surface plasmon resonance‐based assays were established for kinetic/affinity evaluation of antibody–FcγR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant‐made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cell‐derived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant‐produced antibodies.     

Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: APPLICATIONS TO BISPECIFIC MOLECULES AND MONOVALENT IgG

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.     

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild‐type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.     

IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs

Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement.     

Blockade of Fc receptor-mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens

The effect of murine IgG hybridoma antibodies directed against leukocyte antigens on the Fc receptor function of human cells was studied. For this purpose, the specific binding of 125I-labeled monomeric human IgG1 to a macrophage-like cell-line (U-937) was quantitated before and after incubation in the presence of murine monoclonal hybridoma antibodies. Four monoclonal hybridoma antibodies (A1G3, 23D6, 4F2, and 3A 10), each of which binds to different antigens on the surface of U-937 cells, rapidly and potently inhibited the specific binding of labeled IgG1 to these cells. Inasmuch as inhibition was mediated only by IgG antibodies with an intact Fc fragment and antibody activity against surface antigens found on U-937, inhibition appears to have resulted from the formation of a three-component complex composed of antibody bound by its Fab portion to antigen and by its Fc fragment to a Fc receptor. Equilibrium binding studies performed on treated cells confirmed that reduced Fc receptor-mediated binding was due to a reduction in the number of available receptors. Binding studies employing double isotope labeling methods demonstrated that about 0.5 to 1.0 Fc receptor was blocked for each molecule of intact antibody bound to a U-937 cell. Using several techniques, it was shown that most of the monoclonal antibody bound to cells and the Fc receptors blocked by antibody remained on the cell surface despite incubation at 37 degrees C for 3 hr. Thus, the loss of receptor function observed in these experiments was almost exclusively due to reversible receptor blockade rather than receptor internalization or degradation. The antibodies identified in these studies also markedly inhibited Fc receptors on one other human cell line (HL-60) as well as those on normal human peripheral blood monocytes.     

The contribution of constant region domains to the binding of murine IgM to Fc mu receptors on T cells

IgM hybridoma constant region domain deletional mutants were used to investigate the domain requirements for binding of murine IgM to Fc mu receptors (Fc mu R) on normal murine T lymphocytes. Parental Sp 6:18 (mu, kappa; anti-trinitrophenyl) and its mutant proteins or their trinitrophenyl-antigen immune complexes were tested for their ability to inhibit the binding of pentameric IgM to Fc mu R on T lymphocytes. Inhibition was observed with ligands containing multiple copies of the third constant region domain. Inhibition did not occur with ligands missing the third constant region domain. In addition, a battery of rat monoclonal antibodies specific for individual murine IgM constant region domains was tested for the ability to inhibit the binding of pentameric murine IgM to Fc mu R on normal murine T lymphocytes. Total inhibition was observed with the antibodies directed to different epitopes located in C mu 3, but significant inhibition was not observed with antibodies directed to C mu 1, C mu 2, or C mu 4. Studies with domain deletional mutants and anti-domain antibodies have independently provided strong evidence that the C mu 3 domain plays a major role in the binding of IgM to Fc mu R on T lymphocytes and that C mu 1, C mu 2, and C mu 4 are not essential for binding. These studies have also provided evidence that valency and avidity influence the binding of IgM to T lymphocytes that express Fc mu R.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Soluble monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.     

Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.     

Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.     

The two binding-site models of human IgG binding Fc gamma receptors

Fc receptors (FcR) are immunoglobulin-binding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and with their ligands. Recent data on the structure of IgG binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity Fc gamma RI possess one active binding site specific for contact residues that is located at the N-proximal end of the CH2 domain and is able to mediate both binding and signal transfer. The low-affinity Fc gamma RIII has two active binding sites: the CH3 domain-specific site, which mediates only binding; and the CH2 domain-specific site, which is responsible for binding and signaling. Similarly, the low-affinity Fc gamma RII on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc gamma RII on B cells correlates with the cell cycle.