Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human Fc gamma R, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the Fc gamma RI and Fc gamma RII classes but that the YB2/0 antibody was superior in Fc gamma RIII binding. When measuring complexed IgG binding, the difference was 45-fold for Fc gamma RIIIa 158F, 20-fold for Fc gamma RIIIa 158V and approximately 40-fold for Fc gamma RIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with Fc gamma RIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 10(3)-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where Fc gamma RI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only Fc gamma RIII binding and Fc gamma RIII-mediated functions. Relating this to the in vivo studies confirms the importance of Fc gamma RIII in RBC clearance.