Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc C_H2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the C_H2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

A close look at human IgG sialylation and subclass distribution after lectin fractionation

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti‐inflammatory properties depend on sialylation of the N‐glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti‐inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.     

Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization

Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain.     

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF³, and GnGnF⁶ CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.     

Local and global anatomy of antibody‐protein antigen recognition

Deciphering antibody‐protein antigen recognition is of fundamental and practical significance. We constructed an antibody structural dataset, partitioned it into human and murine subgroups, and compared it with nonantibody protein‐protein complexes. We investigated the physicochemical properties of regions on and away from the antibody‐antigen interfaces, including net charge, overall antibody charge distributions, and their potential role in antigen interaction. We observed that amino acid preference in antibody‐protein antigen recognition is entropy driven, with residues having low side‐chain entropy appearing to compensate for the high backbone entropy in interaction with protein antigens. Antibodies prefer charged and polar antigen residues and bridging water molecules. They also prefer positive net charge, presumably to promote interaction with negatively charged protein antigens, which are common in proteomes. Antibody‐antigen interfaces have large percentages of Tyr, Ser, and Asp, but little Lys. Electrostatic and hydrophobic interactions in the Ag binding sites might be coupled with Fab domains through organized charge and residue distributions away from the binding interfaces. Here we describe some features of antibody‐antigen interfaces and of Fab domains as compared with nonantibody protein‐protein interactions. The distributions of interface residues in human and murine antibodies do not differ significantly. Overall, our results provide not only a local but also a global anatomy of antibody structures.     

A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G

We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.     

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1 antibody-antigen interaction on the lymphocyte membrane

In this paper, we characterize the antigen recognized by the monoclonal antibody B73.1 and the modification occurring at the membrane of the positive cells after interaction with the antibody. The B73.1-defined antigen is a protein of 50,000 to 72,000 daltons that is sensitive to pronase but not to trypsin treatment. B73.1 antibody, and its F(ab')2 fragment, directly block, at high concentrations, the binding of IgG antibody-sensitized erythrocytes to the Fc receptors (FcR) of a subpopulation of lymphocytes and neutrophils. B73.1 antibody dissociates rapidly from the positive cells, but concomitant modulation of both B73.1 antigen and FcR is induced when cells are incubated in the continuous presence of antibody or when B73.1 antibody is cross-linked at the cell membrane with an anti-mouse immunoglobulin antiserum. Reaction of lymphocytes with immune complexes also induces modulation of both FcR and B73.1 antigen, without affecting the expression of other antigens on the positive cells. The possibility that the antigen is internalized and digested by the cell after reaction with the antibody is discussed. B73.1 antibody inhibits antibody-dependent cytotoxicity mediated by lymphocytes (K cells) and neutrophils, whereas it does not affect spontaneous cytotoxicity of NK cells. These results suggest the B73.1-defined antigen might be the FcR or a structure closely related to it on K/NK cells.     

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins’ catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.     

Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity.

Recent studies indicate that the herpes simplex virus type 1 (HSV-1) Fc receptor (FcR) can bind antiviral immunoglobulin G by participating in antibody bipolar bridging. This occurs when the Fab domain of an immunoglobulin G molecule binds to its antigenic target and the Fc domain binds to the HSV-1 FcR. In experiments comparing cells infected with wild-type HSV-1 (NS) and cells infected with an FcR-deficient mutant (ENS), we demonstrate that participation of the HSV-1 FcR in antibody bipolar bridging reduces the effectiveness of antibody-dependent cellular cytotoxicity.     

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.     

Analysis of Bacterial Immunoglobulin-Binding Proteins by X-Ray Crystallography

Protein crystallography offers a powerful means of analyzing the molecular mechanisms that underlie the action of bacterial immunoglobulin-binding proteins. Successful approaches used to date involve the isolation of individual IgG-binding domains from the immunoglobulin-binding protein under study and the crystallization of these on their own or in complex with Fc or Fab fragments. Two structures of complexes that have been determined to high resolution by protein crystallography are compared. A single IgG-binding domain from protein A (from Staphylococcus) binds to a human Fc fragment through formation of two α-helices, which bind in the cleft between the CH2 and the CH3 domains. Recognition is mediated by side chains on protein A which interact with conserved side chains on the surface of the antibody, ensuring binding to IgG molecules from different subclasses and species. A similar analysis of the complex of a single IgG-binding domain from protein G (from Streptococcus) with an Fab fragment from mouse IgG1 reveals that the same problem in molecular recognition is tackled in a different way. Protein G binds via an antiparallel alignment of β-strands from the IgG-binding domain and the CH1 domain in Fab: this main chain-main chain interaction is supported by a number of specific hydrogen bonds between the side chains in both proteins. By recognition of a high proportion of main-chain atoms, protein G minimizes the effects of IgG sequence variability in a way that is distinct from that adopted by protein A.     

IgG cooperativity – Is there allostery? Implications for antibody functions and therapeutic antibody development

A central dogma in immunology is that an antibody's in vivo functionality is mediated by 2 independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. However, this view has recently been challenged by reports suggesting allostery exists between the 2 regions, triggered by conformational changes or configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. Here we review the types of cooperativity in IgG molecules by examining evidence for and against allosteric cooperativity in both Fab and Fc domains and the characteristics of associative cooperativity in effector system activation. We investigate the origin and the mechanism of allostery with an emphasis on the C-region-mediated effects on both V and C region interactions, and discuss its implications in biological functions. While available research does not support the existence of antigen-induced conformational allosteric cooperativity in IgGs, there is substantial evidence for configurational allostery due to glycosylation and sequence variations.     

A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2

Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1–3/4GlcNAcβ1–2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca²⁺ in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

No direct association between Ia antigens and Fc receptors on the B cell membrane.

The capping of Ia antigens does not induce redistribution of Fc receptors (FcR) on B lymphocytes. This rules out the possibility of a unidirectional association between Ia and FcR such as has been reported to link Ig and FcR. Ia-capping was achieved with hapten-sandwich antibodies devoid of Fc regions: hapten-conjugated Fab anti-Ia followed by (Fab')2 anti-hapten antibody. Three different immune complex systems were used to label FcR. With fluorescent double labeling, Ia and FcR were readily distinguished. The independent labeling and surface mobility of Ia and FcR are considered in connection with reports of the inhibition of FcR by anti-Ia antibodies.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.     

Study of rabbit IgG, modified with carboxycarbonic anhydride of diethylenetriaminopentacetic acid

The affinity-purified by chromatography on immobilized antigen rabbit IgG was modified with mixed carboxycarbonic anhydride of DTPA which markedly alters the interaction of charged residues in the protein molecule. To study the correlation between the antigen binding activity and the conformational mobility of IgG, the reactivity of modified IgG towards conformational probes targeted at variable and constant IgG domains, was investigated. The antibody against CH2 domains of IgG, staphylococcal protein A and protein antigen ferritin were used as conformational probes. It was found that modification of IgG amino groups entails the global increase in conformational mobility involving the Fab fragments, CH2 and, probably, the CH3 domains of the Fc portion of IgG. Taking advantage of Fab fragments modification it was shown that two processes contribute to the global increase in the conformational mobility of IgG. These processes are: i) stimulation of segmental flexibility and, ii) increase in the mobility within the Fv domains of the Fab fragments.     

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab)₂ fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2AG2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.Abbreviations used in this paper Ig immunoglobulin - FcR receptor for the Fc portion of Ig - TNP trinitrophenyl - Fab antigen-binding fragment - pA Protein A - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - PBS phosphate-buffered saline - BSA bovine serum albumin - SAMIg sheep antimouse Ig - SRBC sheep red blood cells - C complement - FITC fluorescein isothiocyanate - CNBr cyanogen bromide - EA antibody-sensitized erythrocytes