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

Abstract

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...

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

“…The binding affinity of IgG1 to FcgRIIIa is 1.1 mM as determined by SPR, which is consistent with previous reports (0.1 to 10 mM). 22 In contrast, mFc and scFv-mFc did not show any detectable binding to FcgRIIIa. This selective binding pattern was also confirmed by cells expressing Fcg receptors, and there was also no association between mFc and FcgRIIa or FcgRIIbexpressing cells (Fig.…”

Bi-specific Aptamers Mediating Tumor Cell Lysis

“…The low affinity isoform Fc␥RIII␤ (CD16␤) is highly related to CD16␣ and expressed on human neutrophils and eosinophils. Both isoforms can be proteolytically cleaved off cells (25,26), but the prevalent soluble isoform is sCD16␤ (27). The CD16␣ polymorphism Phe-158 to Val-158 enhances its affinity for IgG1 and is associated with improved clinical outcome in tumor patients treated with therapeutical antibodies (28,29).…”

Extravasations and emigration of neutrophils to the inflammatory site depend on the interaction of immune-complex with Fcγ receptors and can be effectively blocked by decoy Fcγ receptors

“…on May 11, 2018. by guest www.bloodjournal.org From to further understand the IC-induced inflammatory pathways leading to neutrophil emigration in mice with intact complement system and Fc␥Rs, we analyzed acute inflammation induced by IC after administration of novel decoy Fc␥R dimers. The dimeric CD16A-Ig fusion protein we used 22,23 does not block complement activation by IC, but maintains its potential to competitively block the interaction of ICs with Fc␥R-expressing inflammatory cells. Our data demonstrate that the interaction of IC with Fc␥R-expressing cells, present at the extravascular site, is critical for the production of complement-independent chemoattractants that trigger the extravasation and emigration of neutrophils during the early phase of the cutaneous Arthus reaction.…”