Mapping the functional topography of Fcγ with monoclonal antibodies: localization of epitopes interacting with the binding sites of Fc receptor on human K cells

Abstract

A panel of monoclonal antibodies (mAb) specific for the C gamma 2, C gamma 3 or inter C gamma 2/C gamma 3 domain epitopes was tested for inhibition of antibody-dependent cellular cytotoxicity (ADCC) specific for anti-D IgG-coated erythrocytes. Significant inhibition of ADCC was demonstrable for some antibodies having specificity for C gamma 2 or C gamma 3 domain epitopes, while others gave no inhibition. Fab fragments of a representative C gamma 2-specific antibody (A55) and C gamma 3-specific antibody (x3a8) retained their inhibitory capacity in lymphocyte-mediated ADCC, but only A55 Fab inhibited monocyte-mediated lysis. Furthermore, the Fab portion of A55 completely abolished the complement-dependent enhancement of ADCC mediated by concanavalin A-stimulated cells, while x3a8 Fab had no effect in this system. On the other hand, x3a8 Fab inhibited the binding of anti-D IgG-sensitized erythrocytes to lymphocytes while A55 Fab did not influence this latter interaction. The results suggest that C gamma 2 domain-FcR interaction is essential for the triggering of lytic process both in lymphocyte and in monocyte-mediated ADCC, while C gamma 3 domain has no role in the latter but is responsible for the appropriate contact between effector lymphocytes and target cells. A site in the region of Lys274 appears to be critical for triggering of both lymphocyte and monocyte-mediated ADCC.

IgG‐Fc‐mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation

“…Fc glycosylation appears to be much less critical for binding of IgG to FcyRIII on human (N)K cells (57) although it has been reported to be essential for triggering of (N)K-cell FcyR(III)-mediated ADCC (45,57). This Immimoiiiflicfll Reviews 163/1998 apparent dissociation between ligand binding and the ability to generate a cellular response is consistent with the previous proposal that FcyRIIIA binding may be mediated through a site within the Cii3 domain but an additional interaction with a CH2 domain site is required for triggering (61).…”

The IgG Binding Site of Human FcγRIIIB Receptor Involves CC′ and FG Loops of the Membrane-proximal Domain

“…The minor differences in the extracellular polypeptide sequences of the Fc␥RIII A and B were shown not to be responsible for the improved binding capacity of the A isoform, since the chimera Fc␥RIIIA/B harboring the extracellular part of the A isoform in the B gene bound hIgG1 complexes and dimers similarly to the Fc␥RIIIB. An additional binding site for Fc␥RIIIA has been proposed in the CH3 domain of IgG (53)(54)(55). Binding curves obtained in our experiments do not refer to the existence of a second binding site on Fc␥RIIIB.…”

Thermodynamics of the Interaction of Human Immunoglobulin E with Its High-Affinity Receptor FcεRI

“…These species interact reversibly in vitro to form a 1:1 complex with an affinity of 1.7 × 10 5 M -1 at 22.0 °C and a ∆C p °of -360 ( 40 cal mol -1 K -1 (29). This interaction appears to be localized to the lower hinge region of IgG 1 -Fc, as shown by site-directed mutagenesis studies, which have shown that residues L234-G237 in the lower hinge (30)(31)(32) and K274-E294 on the proximal bend of Cγ2 (33) are critical for huFcγR recognition (34). In contrast, deletion and sitedirected mutagenesis of the IgE-Fc have implicated several widely separated regions of C 3 in the contact with Fc RI, including the N-terminal linker region analogous to the lower hinge region of IgG (10,21,35), the CD, FG, and EF loops (22), and the AB loop (23).…”