Abstract
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 FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-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 FcγRI 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 FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.
Highlights
For 40 years, human polyclonal anti-RhD antibodies have been used successfully in the prophylactic treatment of haemolytic disease of the foetus and newborn to prevent the immunisation of RhD-negative women by RhD-positive foetal red blood cells (RBC)
Monoclonal anti-RhD antibodies form perhaps the largest group of different antibodies against the same antigen that have been tested in humans. It appears that the most efficient antibodies for RBC clearance are those that give good antibody-dependent cell-mediated cytotoxicity (ADCC) with NK cells [5,6]. This does not necessarily imply that NK cells are involved in RBC clearance but that this assay is a good measure of ability to interact with FccRIIIa
For FccRIIa, FccRIIIa and FccRIIIb, functional polymorphisms are known [26] and testing was performed for two allotypes of each receptor
Summary
Monoclonal anti-RhD antibodies form perhaps the largest group of different antibodies against the same antigen that have been tested in humans It appears that the most efficient antibodies for RBC clearance are those that give good antibody-dependent cell-mediated cytotoxicity (ADCC) with NK cells [5,6]. This does not necessarily imply that NK cells are involved in RBC clearance but that this assay is a good measure of ability to interact with FccRIIIa. Phagocytosis by splenic macrophages is held to be the mechanism of IgG-sensitised RBC destruction but to achieve this by engagement of the high affinity IgG receptor, FccRI, would require displacement of serum IgG, which occupies its binding site under physiological conditions. This could both activate the macrophages directly and promote interactions via FccRI molecules upon dissociation of non-specific IgG from their binding sites
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