Heterogeneity of IgG1 monoclonal anti-Rh(D): an investigation using ADCC and macrophage binding assays (original) (raw)
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Anti-Rh(D) monoclonal antibodies: Study of their in vitro functional properties
Transfusion Clinique et Biologique, 1996
The functional properties {ADCC and phagocytosis} of the anti-RhID } monoclonal antibodies {Mabs) selected for the 3 rd International Workshop were assessed. After sensitization of Rh ~ red blood cells {RBCs), the functional tests were performed by using a colorimetric method. In addition, some samples were selected to study their reactivity against RBCs by a SoI-ELISA.
Blood, 1987
A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number ...
Cellular Immunology, 1988
Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-@ immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supematants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-&$ reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal antihuman IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use ofpolyclonal anti@ reagents to detect these cells has to be carefully evaluated. o 1988 Academic Press, Inc. ' Abbreviations used: EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbcnt assay; FCS, fetal calfserum; Ig, immunoglobulin; RT, room temperature; FITC, fluorescein isothiocyanate; C-10, clone 10; PBS, phosphate-buffered saline.
Transfusion Clinique et Biologique, 2002
Sixty four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (FcγR)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit FcγRI, FcγRII or FcγRIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be FcγRI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fcγ receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through FcγRI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through FcγRIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through FcγRIII. Haemolysis via FcγRIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only FcγRI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through FcγRI and FcγRIII while IgG2 was inhibited by Mabs to both FcγRII and FcγRIII, suggesting a variety of FcγR are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.
British Journal of Haematology, 2008
A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD + red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac Ò ). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD + RBCs. Autologous RhD + RBCs were sensitized with either Rhophylac Ò or R297 at three different coating percentages (25, 12AE5 and 6AE25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6AE25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FccRIII binding and increased ADCC.
Functional in vitro studies of recombinant human immunoglobulin�G and immunoglobulin�A anti-D
Transfusion, 2007
The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and nonplasma-derived source of antibodies for Rhesus prophylaxis is needed. STUDY DESIGN AND METHODS: Recombinant human immunoglobulin G (IgG)1, IgG2, IgG3, IgG4, IgA1, and IgA2 anti-D with the same variable region were expressed in Chinese hamster ovary cells. The effector functions of these antibodies were assessed by an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and a chemiluminescence (CL) method for detection of respiratory burst. RESULTS: In the ADCC assay, IgG1, IgG3, and IgA1 did the best and were as active as a currently used prophylactic polyclonal anti-D. IgG4 and IgA2 were moderately active, whereas IgG2 was not active. In the CL assay, IgG1 and IgG3 were active but much less so than a currently used prophylactic polyclonal anti-D. For some effector cell preparations, IgG4 was active in the CL assay, whereas IgG2, IgA1, and IgA2 were not. A mixture of IgG1 and IgG3 showed a synergistic effect in the CL assay and did as well as the prophylactic polyclonal anti-D in ADCC and CL. Mixtures of IgA1 and either IgG1 or IgG3 showed no synergistic effect. CONCLUSION: A mixture of recombinant human IgG1 and IgG3 anti-D could be of value in future Rhesus prophylaxis.
Human Monoclonal Antibodies to Rh o (D
Vox Sanguinis, 1987
Abstract. B lymphocytes from Rh negative donors with serum anti-D antibodies were isolated and fused with the mouse-human heteromyeloma, SBC-H20, to produce hybridomas secreting IgM or IgG1 human monoclonal antibodies to D antigen. The IgM antibody in hybridoma supernatant agglutinates all normal D positive cells at the immediate spin phase of reactivity. Using concentrated IgM hybridoma supernatant of approximately 50 μg/ml, Du cells were also agglutinated. The IgG1 antibody reacts by indirect hemagglutination with all D and Du cells. Against Rh mosaics, different reactivity was noted for each antibody. Furthermore, D positive cells precoated with the IgG1 antibody inhibit the IgM direct hemagglutination, suggesting that the antibodies identify closely associated epitopes. These human monoclonal antibodies will be useful diagnostic reagents and, ultimately, should be useful in the prevention of Rh hemolytic disease of the newborn.
Flow cytometric evaluation of non anti-RH1(D) monoclonal Rh antibodies
Transfusion clinique et biologique : journal de la Société française de transfusion sanguine, 1996
IgG non anti-RH1(D) monoclonal Rh antibodies were evaluated by flow cytometry. The values obtained with these antibodies were less strong than those obtained with anti-RH1(D) antibodies. For a significant number of antibodies, the signal was not high enough to give reliable results for the antibody specificity. Despite these drawbacks, flow cytometry was an efficient tool to appreciate the variation of reactivity by different antibodies with normal or variant cells. These variations were not always obvious by serological means.
PLoS ONE, 2014
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 YB2/0produced 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 FccR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FccRI and FccRII classes but that the YB2/0 antibody was superior in FccRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FccRIIIa 158F, 20-fold for FccRIIIa 158V and approximately 40-fold for FccRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FccRIIIa. 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 FccRI 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 FccRIII binding and FccRIIImediated functions. Relating this to the in vivo studies confirms the importance of FccRIII in RBC clearance.