Mapping epitopes of human Fc gamma RII (CDw32) with monoclonal antibodies and recombinant receptors

Abstract

Fc gamma RII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1 gamma and D2 gamma), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human Fc gamma RII mAb (8.2, 8.7, 8.26, and 7.30) are detailed, and the mAb are compared with two defined CDw32 mAb, IV.3 and CIKM5. Reactivity of all mAb with Fc gamma RII was demonstrated by (a) specific binding to Fc gamma RII+ L cells (produced after transfection of L cells with human Fc gamma RIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to Fc gamma RII+ L cells, and (c) immunoprecipitation and SDS-PAGE, which detected a 42-kDa protein on K562 and U937 cells and a single 45-kDa protein on Fc gamma RII+ L cells. The mAb were able to detect different forms of Fc gamma RII, by flow cytometry, on Daudi cells (8.7 and 7.30) and U937 cells (8.2, IV.3, and CIKM5); 8.26 stained Daudi cells with intermediate fluorescence and U937 cells with the highest fluorescence, relative to the remaining mAb. Binding to transiently expressed isoforms of Fc gamma RII (a and b1) and four allelic variants of Fc gamma RIIa in COS-7 cells did not distinguish the mAb epitopes. Further mapping of the mAb epitopes was determined by (a) EA inhibition assays, (b) mAb blocking studies, and (c) the binding of the mAb to segments of human Fc gamma RIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1 gamma linked to domain 2 of Fc epsilon RI or domain 1 of Fc epsilon RI linked to D2 gamma were produced by exchanging homologous, but antigenically different, regions of Fc gamma RIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of Fc gamma RII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1 gamma and D2 gamma distant from the IgG Fc binding site, inasmuch as F(ab')2 fragments of 8.2 and CIKM5 do not inhibit the binding of EA to Fc gamma RII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2 gamma and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to Fc gamma RII. Thus, all mAb that inhibit the binding of EA map totally to D2 gamma; it is likely the IgG Fc binding region is also contained in D2 gamma.