Monoclonal anti-D Antibodies Research Articles

Measurement of the affinity of anti‐D in the serum of immunized mothers and in immunoglobulin preparations with unlabeled antibodies

Few data are available on the affinity of maternal anti-D responsible for hemolytic disease of the fetus and the newborn (HDN) and of anti-D used for the prophylaxis of that disease. A method was recently described to measure the affinity (K(a)) of untagged anti-D monoclonal antibodies (MoAbs). In this work, the same method was applied to determine the K(a) of polyclonal anti-D. O R(1)r red blood cells (RBCs) were sensitized with increasing concentrations of native anti-D in serum samples from immunized mothers and donors and in RhIG preparations. At equilibrium, the amount of anti-D bound to RBCs was measured by enzyme-linked immunosorbent assay. Scatchard and Langmuir equations were used to determine Ka. The experimental data fitted well with the Scatchard equation (mean r2=0.95) but a better correlation was observed with the Langmuir equation (mean r2=0.99). The mean Ka of anti-D in 11 maternal serum samples, in 6 immunized donors, and in 5 lots of RhIG were 5.6x10(8) per M (from 2.8x10(8) to 12x10(8)/M), 3.9x10(8) per M (from 1.5x10(8) to 6.8x10(8)/M), and 3.4x10(8) per M (from 3.1x10(8) to 4.2x10(8)/M), respectively. The comparison of anti-D affinity in 5 cases of HDN with fetal anemia and in 6 cases of HDN with postnatal anemia showed no significant difference. The method previously described for anti-D MoAbs was applied to polyclonal anti-D present in the serum of immunized subjects and in immunoglobulin preparations. The experimental data fitted well with the Langmuir equation, and the affinity of polyclonal of anti-D was measured with accuracy.

Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice

The prophylaxis of the hemolytic disease of the newborn requires significant amounts of plasma-derived polyclonal human anti-D. Because of procurement problems, there is a growing interest in replacing plasma-derived anti-D by in vitro-produced human monoclonal anti-D. Hundreds of monoclonal anti-D have been prepared, but the selection of the most potent for in vivo use is difficult because it cannot be predicted by in vitro characterization. This study evaluated the possibility of using nonobese diabetic/severe combined immunodeficient (NOD-scid) mice for the in vivo evaluation of human monoclonal anti-D. Human red blood cells (RBCs) were found to circulate normally in the blood of NOD-scid mice previously injected with a physiologic amount of human immunoglobulin G (10 mg). The addition of a small amount of anti-D (1 to 5 microg) resulted in the clearance of Rh D(+) RBCs within 4 hours. The comparative testing of 8 monoclonal anti-Ds showed a wide range of potency (15% to 87%) relative to plasma-derived polyclonal anti-D. There was no strong correlation between the in vivo potency index and the immunoglobulin G isotype, affinity, or fine specificity of the antibodies. These results show the usefulness of NOD-scid mice for the initial in vivo screening of human monoclonal anti-D before testing the most active antibodies in clinical trials done in human volunteers.

Section 1B: Rh flow cytometryCoordinatorˈs report.Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated F ab fragment goat anti-human IgG (H+ L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal ‘standard RBC’ throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for D VI type III, followed by D VII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.

Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti-D antibodies (mAbs) has shown that weak D red cells display type-specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment. We amplified DNA from four Japanese weak D samples using the polymerase chain reaction (PCR), and directly sequenced the amplified DNA. Using site-directed mutagenesis, we constructed three vectors expressing mutant RHDs-- G212C, V270G (weak D type 1) and G358A (type 2)--in K562 cells. The expression of RhD antigens was examined by flow cytometry using mAbs. A new mutation resulting in a conversion at amino acid residue 212 (Gly to Cys) was detected in a Japanese weak D sample. K562 cells transduced with mutant RhD cDNA reacted weakly in a type-specific manner with mAbs. The mutations--G212C (new weak D type), V270G (weak D type 1) and G358A (type 2)-- in transmembranous regions had obvious effects on the D epitopes recognized by mAbs. The results of this study provide direct evidence that these mutations can account for weak D phenotypes.

Fc receptor blockade and immune thrombocytopenic purpura

Inhibition of antibody-coated platelet destruction in patients with immune thrombocytopenic purpura (ITP) is a well-known mechanism of treatment effect. A number of interventions that would ameliorate the thrombocytopenic effect of ITP patient plasma when infused into normal recipients were demonstrated in 1965. Subsequently, the antibody-coated chromium-labeled red blood cell clearance study was developed to allow direct in vivo assessment of Fc receptor (FcR) blockade. This was first demonstrated for corticosteroids but more extensive investigation began with the study of intravenous infusions of gammaglobulin (IVIg). The unequivocal demonstration of FcR blockade following IVIg initiated novel approaches. One involved the infusion of a monoclonal anti-FcRIII ligand-blocking antibody into patients with refractory ITP. The efficacy of this treatment demonstrated that FcR blockade was not an epiphenomenon but rather an important mechanism of the increase in the platelet count in patients with ITP. Confirmation of its importance was obtained from the infusion of intravenous (IV) anti-D and the use of the isolated Fc piece of IgG. Recent studies have begun to explore the possibility of a monoclonal anti-FcRI and monoclonal anti-Ds. In summary, FcR blockade is an important mechanism of treatment effect in patients with ITP. Cytokine release as a consequence of this interaction and other immunomodulatory effects have only begun to be studied.

The estimation of fetomaternal haemorrhage

Prepared by a working party of the BCSH Blood Transfusion (Chairman P. Kelsey) and General Haematology (Chairman J. T. Reilly) Task Forces Working party: J. F. Chapman, B. J. Bain, S. C. Bates, S. M. Knowles, K. H. Shwe, J. Parker-Williams, L. Robson and S. C. Robson Blood Transfusion Task Force: M. Bruce, J. F. Chapman, J. Duguid, S. M. Knowles, M. F. Murphy, L. Williamson, I. Cavill and J. K. Wood Haematology Task Force: R. J. Amos, B. J. Bain, C. Chapman, K. Hyde, E. Matutes, J. Parker-Williams, I. Cavill and J. K. Wood

A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblotting: Apoil PA, Reid ME, Halverson G, et al. Br J Haematol 98:365–374, 1997

A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblotting: Apoil PA, Reid ME, Halverson G, et al. Br J Haematol 98:365–374, 1997

Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing.

An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG. In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination.

Absence of expression of RhD by human trophoblast cells

Our purpose was to determine whether the RhD gene is expressed in trophoblast at any stage of gestation. Trophoblast and fetal tissue were obtained from 18 pregnancies at 8 to 40 weeks' gestation. Deoxyribonucleic acid and ribonucleic acid were extracted from trophoblast. Complementary deoxyribonucleic acid was synthesized from ribonucleic acid, and reverse transcriptase-polymerase chain reaction was performed using primers specific for the RhD gene. Deoxyribonucleic acid was extracted from fetal tissue to determine the fetal RhD status by means of polymerase chain reaction. Antigen expression was also sought by analytic cytometric analysis (flow cytometry and immunocytochemistry) using a monoclonal anti-D antibody. Trophoblast was studied from various combinations of RhD-positive and RhD-negative fetuses (on deoxyribonucleic acid) from mothers to find no RhD gene expression in any sample. Flow cytometry and immunocytochemistry confirmed this by demonstrating no RhD antigen sites on trophoblast cells. Contrary to a previous report, we conclude that the RhD gene is not expressed in human trophoblast in any trimester.

There is no relationship between the activities of mono- and polyclonal antirhesus immunoglobulinsin vitro andin vivo

Monoclonal anti-D antibodies induce lysis of rhesus-positive erythrocytes in the antibody-dependent cytolysis test with blood mononuclear cells, the activity of the best antibodies being 10–15 times higher than of polyclonal IgG. The activities of anti-D antibodies in the cytolysis test with monocytes and lymphocytes varied: only one monoclonal antibody was highly active in both tests, whereas polyclonal IgG caused no lysis of erythrocytes in the cytolysis test with monocytes but was active in the same test with lymphocytes. Monoclonal antibodies intravenously administered to rhesus-negative recipients after transfusion of rhesus-positive erythrocytes stimulated their clearance, but its rate was slower than with polyclonal antibodies. Erythrocytes sensitizedin vitro with monoclonal antibodies were completely removed from circulation within 3 h. It is not clear the results of which of the studied tests correlate with the capacity of anti-D immunoglobulins to block rhesus sensitization.

Role of Fc gamma RIIa (CD32) in IgG anti-RhD-mediated red cell phagocytosis in vitro.

To test the role of Fc gamma RIIa in IgG anti-RhD-mediated phagocytosis three IgG1 and two IgG3 human monoclonal anti-D antibodies were tested for ability to mediate binding/phagocytosis of cDE/cde and -D-/-D- red cells by Fc gamma RIIa-R131 and Fc gamma RIIa-H131 cDNA-transfected 3T6 fibroblasts. Both IgG3 monoclonal antibodies brought about -D-/-D- cell interaction with IIa-transfected fibroblasts, while only one of them, Og3, mediated binding of cDE/cde targets. Although Fc gamma RIIa expression was three times greater on IIa-R131 than on IIa-H131 fibroblasts, the latter bound significantly more Og3-coated cDE/cde- and IgG3 anti-D-sensitized -D-/-D- cells, respectively, than the former effectors and showed some phagocytosis of the -D-/-D- targets. IgG1 anti-D antibodies were inactive in mediating red cell interaction with the fibroblasts. Moreover, monoclonal anti-Fc gamma RII IV.3 partially inhibited the phagocytosis by adult or fetal monocytes of Og3-sensitized cDE/cde cells. Fc gamma RIIa-H/H131 monocytes exhibited higher phagocytic indices towards these targets than monocytes of other IIa allotypes. The results indicate that Fc gamma RIIa can participate in the phagocytosis of red cells coated with IgG3 anti-D; in this case the allotype of the receptor will modify the extent of red cell destruction.

Frequency of partial D phenotypes in the south western region of France.

The aim of this study was to estimate the frequencies of partial D phenotypes among weak D (D14) blood samples. During one year, red blood cell samples with D14 phenotype were tested by the indirect antiglobulin test (IAT) using a panel of six anti-D monoclonal antibodies (Mabs). The panel of Mabs was selected for its capacity to define patterns of reactivity specific to the most frequent categories of partial D phenotypes. Among 475 D14 samples, 16 partial D phenotypes were identified (2 DIIIb, 3 DIVa, 4 DIVb et 7 DVI) corresponding to 3.36% of the D14 population. A strategy for systematic screening of partial D phenotypes is discussed in relation to the results of this study.

Human Rh monoclonal antibodies: assessment of functional activity by chemiluminescence and RhD antibody quantitation.

In vitro cellular assays have been described which are capable of evaluating the interactions between sensitised red cells and monocyte or K cells. The chemiluminescence assay (CL) has several advantages over other cellular assays used to assess functional activity. The CL assay unlike the ADCC assays does not require the use of radioisotopes and therefore can be easily integrated into the work of a Reference Serology laboratory. The CL assay is an objective test and not labour intensive which is the main criticism of the monocyte monolayer assay. Seventy-four monoclonal anti-Ds and 29 other Rh specificities have been evaluated by a CL assay. The use of the chemiluminescent response produced by erythrophagocytosis of sensitised red cells has been shown to correlate well with the in vivo response to red cells sensitized with polyclonal IgG antibodies. This study aimed at investigating whether the CL assay could identify and differentiate monoclonal antibodies that are capable of eliciting a response from human monocytes. Poor correlation was obtained between the CL assay results and anti-D quantitation (r = 0.236). The chemiluminescence assay discriminated between anti-D's with high quantitation levels but low predicted functional activity and anti-Ds of low quantitation levels which produced elevated CL responses. Only 3 of the 29 non-Rh D specificities tested produced a response in the CL assay emphasising the importance of specificity in the functional activity of monoclonal antibodies. The demonstration of significant differences in the functional capabilities of monoclonal antibodies has important implications for reviewing the possible use of monoclonal anti-D preparations for Rh immune prophylaxis and highlights the requirement for factors other than the antibody concentration to be examined.

Monoclonal antibodies directed against human Rh antigens in tests with red cells of non-human primates.

Human anti-D (Rho) monoclonal antibodies (Mabs) of the IgG (70) and IgM (27) classes were tested with red blood cells (RBCs) of various non-human primates, from anthropoid apes to New World monkeys. Significant differences in reactivity were observed among antibodies of two classes depending on taxonomic position of primate animals. Only IgM Mabs gave positive reactions (9 out of 18 Mabs) with blood of Old World monkeys. Allotypic reactions with RBCs of African apes were produced by a majority of IgG Mabs but by very few IgM reagents, most of the latter reacting with RBCs of all chimpanzees and all gorillas tested. Eight out of 70 IgG anti-D defined chimpanzee polymorphisms related to chimpanzee Rc antigen which is the chimpanzee counterpart of human D antigen. Most of IgG anti-D Mabs (61/70) were found specific of Dgor antigen (gorilla counterpart of human antigen D). Most of anti-D which were found negative with all chimpanzee RBCs were also negative with human DIVb RBCs and most of anti-D which agglutinated human DIVb RBCs were positive with some or all chimpanzee blood samples. Differences among Mabs evidenced in tests with non-human primate RBCs reflect the complexity of the immune reactions to the human D antigen. The results obtained with anti-Rh Mabs of specificities other than D confirmed that chimpanzee, gorilla and gibbon express c-like epitopes and that antigens C, E, e are absent in non-human primates.

Reactivity of 62 Rh MAbs with weak and variant antigens.

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated DIV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with DVI and Rh 33 red blood cells. The CwCw sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated Cx samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.

Evaluation of monoclonal anti-D reagents using D variant cells.

Monoclonal anti-D antibodies submitted to the Third Monoclonal International Workshop were evaluated against a number of D variant cells using standard serological techniques. The monoclonal antibodies were able to discriminate between the cells of Categories Va, VI and DFR but not Category III cells. Cells within each category did not give any aberrant results. The Rh:33 cells behaved as normal Rh(D) positive cells.

ADCC activity of monoclonal anti-D antibodies.

ADCC activity of monoclonal anti-D antibodies.

Reactions of anti-D monoclonal antibodies with rhesus D variant cells.

Reactions of anti-D monoclonal antibodies with rhesus D variant cells.

The glycosylation of red cell autoantibodies affects their functional activity in vitro.

Factors governing the functional activity of red cell autoantibodies are poorly defined. Here we report the presence of qualitative differences in the glycosylation of IgG autoantibodies which affect in vitro interactions with Fc gamma RIII. The following antibodies were affinity-purified by adsorption and elution from normal red cells: IgG eluted from the red cells of 27 haemolysing or non-haemolysing patients, anti-D in sera from 11 pregnant women, and IgG1 and IgG3 human monoclonal anti-D. Monoclonal antibodies with differing levels of agalactosyl IgG were produced by culturing cell lines at high or low cell density. The % IgG with oligosaccharides lacking terminal galactose residues (agalactosyl IgG) of antibodies was designated as low, medium or high according to their reactivity with a monoclonal antibody to terminal N-acetylglucosamine. Fc gamma RIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cells sensitization. All eluates containing allo-anti-D were lytic (range 74-100%). In contrast, lysis by autoantibodies varied from 0 to 100%; 11/13 eluates from red cells of haemolysing patients promoted > 5% lysis compared to 2/7 eluates from red cells of non-haemolysing patients (P < 0.02). The ability of autoantibodies to promote K-cell-mediated red cell lysis correlated inversely with their level of agalactosyl IgG (r = -0.56, P < 0.01, n = 23). Further, monoclonal anti-D antibodies with very low levels of agalactosyl IgG were comparatively more lytic than the same antibodies containing more agalactosyl IgG. Analysis of the ratio of kappa:lambda light chains suggested that autoantibodies from 6/19 patients were monoclonal or oligoclonal in nature. The data indicate that IgG red cell autoantibodies from different patients are functionally heterogenous, and that this may be due, at least in part, to qualitative differences in the Fc region glycosylation reflected by differences in the proportion of agalactosyl IgG. This heterogeneity is consistent with the clonally-restricted nature of the autoantibodies in some patients.

Development and evaluation of a competitive radioimmunoassay using monoclonal antibodies against Rh D for determining anti‐D potency of clinical grade immunoglobulin preparations

A competitive radioimmunoassay using monoclonal anti-D antibodies has been developed for measuring anti-D activity in prophylactic immunoglobulin preparations. The assay is totally specific for anti-D, reproducible and gives potency estimates comparable to those determined using autoanalyzer methodology. The assay also provides a means for comparing different batches of monoclonal anti-D preparations for red-cell binding activity.