Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.

Chappel, M. S.; Isenman, David E.; Everett, Margaret; Xu, Yuanyuan; Dorrington, Keith J.; Klein, Michel

doi:10.1073/pnas.88.20.9036

Cited by 119 publications

(84 citation statements)

References 26 publications

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“…Mutations in the Fc domain of hCD16a-Ig are L267F, L268E, G270A, and A363T (numbered as in accession number AAH69020.1). These mutations were shown to abolish the binding of Fc␥Rs (18,19). The anti-CD16 nonblocking monoclonal antibody (mAb) 214.1 (murine IgG1) was a generous gift from Dr. Howard Fleit (State University of New York, Long Island).…”

Section: Methodsmentioning

confidence: 99%

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Jiang²,

Nagarajan

et al. 2007

Journal of Biological Chemistry

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Binding of pathogen-bound immunoglobulin G (IgGFc ␥ receptors (Fc␥Rs) 7 are a family of cell surface glycoproteins with varying affinities for the Fc region of immunoglobulins G (IgG). Three classes of human Fc␥Rs have been described: Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16), which are widely distributed in hematopoietic cell lineages. These include at least 12 different isoforms many of which are polymorphic. For example, CD16 has two isoforms, a and b, that differ by six amino acids in the extracellular domain and the presence (CD16a) or absence (CD16b) of the transmembrane and cytoplasmic domains (1).The IgG-Fc␥R interaction-mediated binding of antibody-opsonized pathogens to leukocytes is a key event by which antibody effector functions are initiated. Kinetic rates and binding affinity are critical determinants of an IgG-Fc␥R interaction, as they control how likely and how rapidly such binding will occur, how many bonds will be formed, and how long the bonds last. In addition, it has been hypothesized that these parameters are related to the signaling events following the initial binding (2-4). Using a micropipette method, we have measured the kinetic rates and binding affinities of several IgG-Fc␥R interactions when both interacting molecules are anchored on apposing surfaces. We found that in this assay the half-lives of these IgG-Fc␥R bonds are in the order of seconds (5-9).In addition to interaction parameters of surface-linked molecules, the kinetic rates and affinities of Fc␥Rs for soluble IgG are also of interest, because in vivo interactions of immobilized IgG to leukocyte Fc␥Rs are subject to competitive binding of soluble antibodies in sera. Of the three Fc␥Rs, CD64 binds to monomeric IgG with high affinity (K d ϳ tens of nM) (10), CD32 and CD16b are of low affinities (K d , several and several tens of M, respectively) (6, 11, 12), and CD16a is considered as an intermediate affinity receptor (K d ϳ hundreds of M) for monomeric IgG (6,10,12). In addition to the above results, which were obtained by conventional assays using Fc␥R-expressing cells, there were two studies using the surface plasmon resonance (SPR) technology and soluble Fc␥Rs. Galon et al. (13) reported half-lives of the order of 10 min and equilibrium dissociation constants of several micromolar for sCD16b NA2 binding to human (h) IgG1 and IgG3. By comparison, Maenaka et al. (14) found a much faster off-rate (half-lives of the order of seconds) but similar K d for IgG-sCD16b NA2 binding. Here, we report kinetic and affinity measurements of a soluble dimeric human Fc␥RIIIa (sCD16a) interacting with various IgG ligands in both monomeric and multimeric forms using SPR. We found that sCD16a interacted with monomeric rabbit (Rb) IgG, hIgG, hIgG1, and hIgG3 with on-rates of 1.8 ϫ 10 4 , 6.5 ϫ 10 3 , 8.2 ϫ 10 3 , and 1.1 ϫ 10 4 M Ϫ1 s Ϫ1, off-rates of 1.9 ϫ 10 Ϫ2 , 4.7 ϫ 10 Ϫ3 , 5.7 ϫ 10 Ϫ3 , and 5.9 ϫ 10 Ϫ3 s Ϫ1 , and equilibrium dissociation constants of 1.1, 0.72, 0.71, and 0.56 M, respectively. sCD16a bound with aggregated li...

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Section: Methodsmentioning

confidence: 99%

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Jiang²,

Nagarajan

et al. 2007

Journal of Biological Chemistry

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“…The key amino acid residues in the Fc region that interact with Fcγ receptors (CD64 for FcγRI, CD32 FcγRII and CD16 FcγRIII), complement C1q and neonatal FcRn receptors have been determined. Earlier work using a domain swapping approach has defined regions that are required for Fcγ receptor (~230-238 amino acids 11 according to the EU numbering system) and complement binding (292-340 amino acids 12 ). Further studies have identified individual amino acid residues that interact with Fcγ receptors and complement C1q protein.…”

Section: Introductionmentioning

confidence: 99%

IgG2m4, an engineered antibody isotype with reduced Fc function

Forrest

Moore

et al. 2009

mAbs

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“…Several results pointed out the importance of residues 233-239 in the human system [8,9,11,12,24,25] [8]. Based on the threedimensional (3D) structure of the human Fc + RIIb, the interdomain cleft between the CH2 and CH3 domain of IgG was suggested as the Fc + RIIb binding site [28].…”

Section: Discussionmentioning

confidence: 99%

“…Hence, monomeric IgG present in the circulation does not trigger responses mediated by Fc + RI, Fc + RII or Fc + RIII [4]. Tryptophan residues in the first domain of all Fc + R play an important role in forming a hydrogen bridge with the ligand [6], and both the lower hinge and several residues in the CH2 domain of IgG were shown to participate in the interaction [6][7][8][9][10][11][12][13][14]. Glycosylation of IgG may also influence binding to certain Fc + R isoforms [15,16].…”

Section: Introductionmentioning

confidence: 99%

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

et al. 2004

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Receptors specific for the Fc part of IgG (Fc + R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc + R may result in autoimmunity. Thus, the modulation of IgG-Fc + R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc + RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P 234 and S 298 were synthesized and used in binding and functional experiments. Binding of the peptides to Fc + R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T 256-P 271 complexed by avidin exhibited functional activity; they induced Fc + RIIb-mediated inhibition of the BCR-triggered Ca 2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF- § and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc + RII-binding part of IgG1.

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Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.

Abstract: To characterize the region on human IgG1

Cited by 119 publications

References 26 publications

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

IgG2m4, an engineered antibody isotype with reduced Fc function

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

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