Mechanisms of Red Cell Destruction Mediated by Non-Complement Binding IgG Antibodies: the Essential Role in Vivo of the Fc Part of IgG

F(ab')2G anti-D was prepared by limited digestion of IgG anti-D with pepsin. Conditions of digestion were chosen in order to obtain a nearly complete conversion of the anti-D antibody molecules as tested by immunochemical and serological techniques. The F(ab')2G anti-D was not capable of inducing adherence of rhesus D positive red cells to monocytes in vitro or of eliminating such cells in vivo in normal volunteers. These findings are compatible with a role of the Fc-receptor-mediated adherence to cells of the macrophage system in vivo in the destruction of erythrocytes by non-complement binding IgG antibodies.     

Human Monoclonal Anti-D with Reactivity against Category DVI Cells Used in Blood Grouping and Determination of the Incidence of the Category DVI Phenotype in the DU Population

B-lymphoblastoid cell lines transformed by Epstein-Barr virus were produced from cells obtained from a hyperimmunised donor with serum anti-D activity against category DVI red cells and enriched for this activity by rosetting with category DVI red cells. Three clones produced IgG1 anti-D and had stable cell growth and continuous secretion of antibody in prolonged culture. The monoclonal antibodies reacted with category DVI red cells, when assessed manually and in an automated blood grouping system, and are useful blood grouping reagents for the detection of the category DVI phenotype. Using a radiometric technique, the number of antibody molecules bound to category DVI red cells from 5 individuals was estimated to range from 2,800 to 11,200 per cell. Five percent of blood donors classed as Du in the south western region were found to have the category DVI phenotype.     

An in-vitro assessment of the functional activity of monoclonal anti-D

The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.     

Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fcγ receptors

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti-D and IgG2 anti-A were isolated by affinity purification from an unusual anti-D serum (DEL) and anti-A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti-D in in vitro functional assays of the interaction between IgG-coated red cells (EA-IgG) and cells bearing IgG Fc receptors (FcγR). Dimeric IgG2 anti-D bound efficiently to cell lines transfected with FcγRIIa-H131, an allotypic form of FcγRIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, -D-phenotype red cells coated with IgG2 anti-D did not form rosettes with these cells, whereas EA-IgG2 anti-A and EA-IgG1 and EA-IgG3 anti-D effectively formed rosettes with these transfectants at the same sensitization level (100 000 molecules IgG/red cell). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, lysis of EA-IgG2 anti-A was mediated via FcγRIIa, whereas lysis of EA-IgG1 and EA-IgG3 anti-D was mediated via FcγRI or FcγRIII; EA-IgG2 anti-D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG–FcγR interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti-D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of FcγRIIa-H131 to the FcγR recognition site on the relatively inflexible IgG2 anti-D, but not to that of IgG1 or IgG3 anti-D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red-cell-bound IgG2 anti-D and IgG2 anti-A with cells bearing Fcγ receptors can occur.     

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display

BackgroundRhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor’s donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display.Material and methodsHaemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count.ResultsOf 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity.DiscussionBFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.     

Ability of monoclonal anti-D antibodies to promote the binding of red cells to lymphocytes, granulocytes and monocytes

Red cells sensitized with IgG1 or IgG3 monoclonal anti-D antibodies were used in rosette assays with human lymphocytes, monocytes and granulocytes. With all three cell types. IgG3 antibodies promoted a greater degree of rosette formation than IgG1 antibodies. Monocytes required a minimum of about 0.5 x 10(3) IgG3 molecules per red cell for rosette formation, and granulocytes and lymphocytes required around 1 x 10(4) IgG3 molecules per red cell. Approximately 80% of monocytes and granulocytes and 10% of lymphocytes were capable of rosette formation. These results are consistent with differences in the number and affinity of Fc receptors on different leucocytes. When compared with previous data these results suggest that binding of monocytes to monoclonal anti-D sensitized red cells is very similar to that of red cells sensitized with polyclonal antisera. Lymphocytes and granulocytes, however, appear to bind less well to red cells sensitized with certain monoclonal antibodies than with polyclonal antibodies. These findings may be of relevance to the prophylactic use of monoclonal anti-D antibodies.     

流式细胞术检测低效价Rh抗体的研究

目的:建立流式细胞仪检测Rh血型低效价抗体的方法。方法:将不同效价IgG抗-D试剂与标准化人源Rh(D)阳性红细胞混合,加入异硫氰基荧光素标记的鼠抗人IgG二抗,用流式细胞仪检测样本抗-D抗体含量。检测结果与抗球蛋白法进行对比。结果:2种方法对于抗-D抗体水平变化趋势的检测结果一致,但流式细胞术可检测到128倍和256倍稀释的低效价抗体,其特异性、灵敏性优于抗球蛋白法。结论:流式细胞术可以用于低效价IgG抗-D抗体的检测,且能将其定量,效果优于传统方法。     

Production of Direct Agglutinins by the Combination of Incomplete Antibodies to Protein A-Bearing Staphylococcus aureus Cells

Abstract: A novel method is described for the production of saline-reacting antibodies from 'incomplete' blood grouping sera. IgG anti-D is combined to protein A-bearing Staphylococcus aureus (SA), forming a polyvalent anti-D assembly. The SA-anti-D has a high titre in saline and is stable on 4°C storage and lyophilisation. The material was successful in Rh(D) typing cells sensitised in vitro with various antibodies. Saline-reacting SA-anti-Kell and SA-anti- were also produced.     

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

Summary. 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 FCγRIII. 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 IgGl and IgG 3 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. FcγRIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cell 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 κ:λ 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.     

Influence of syngeneic monoclonal anti-idiotypic antibodies to murine monoclonal antibodies against tumour-associated antigens on the biodistribution of their target antibodies and their fragments

The effect of syngeneic anti-idiotypic (anti-id) antibodies on the biodistribution of three murine monoclonal antibodies (mAb) against human tumour-associated antigens, and also on that of their fragments, has been examined in mice using, as a model system, purified anti-id mAb against three different target mAb. With the IgG2b mAb NCRC-2, pretreatment of mice 24 h previously with its IgG1 anti-id mAb NCRC-60 caused clearance of subsequently administered NCRC-2. With the univalent Fab/c fragment of NCRC-2 there was little effect, even with anti-id to Fab/c pretreatment ratios of 201, although immune complexes were present in the circulation. With Fab of NCRC-2, anti-id mAb prolonged blood survival by reducing renal clearance, immune complexes surviving in the circulation. With the IgG1 mAb NCRC-23, immune complexes formed in vivo with the IgG2b anti-id mAb NCRC-59, but with only little hepatic clearance. With the Fab and F(ab)₂ fragments of NCRC-23, blood survival was increased in mice pretreated with anti-id mAb, and with Fab this was clearly due to reduced renal clearance. The third mAb, the IgG3 NCRC-48, formed complexes with its IgG2a anti-id mAb NCRC-62, but these survived in the circulation with no accelerated clearance of the target mAb. These results are different from those previously seen with endogenous anti-id responses. They indicate the diversity of effects that anti-id mAb can have on the biodistribution of their target mAb, and emphasise the difficulty of using such anti-id mAb to modulate the pharmacokinetics of target mAb.Abbreviations mAb monoclonal antibody - anti-id anti-idiotypic - PBS phosphate-buffered saline, pH 7.2 - T:B tumour to blood ratio - AUC area under curve of plot of surviving radiolabel against time     

Human monoclonal anti-D antibodies: II. THE RELATIONSHIP BETWEEN IgG SUBCLASS, Gm ALLOTYPE AND Fc MEDIATED FUNCTION

Eight monoclonal antibodies (mabs) to the Rh antigen D produced by Epstein-Barr virus transformed B-lymphoblastoid cell lines from two individuals have been compared for their behaviour in in vitro cell-mediated assays. Three IgG1 Glm(1,17) and two IgG3 G3m(21) mabs from one donor and three IgG1 Glm(3) mabs from another were used. IgG3 anti-D mabs induced greater adherence and phagocytosis of sensitized red cells by U937 monocytes than IgG1 anti-D mabs or the polyclonal anti-D. Minimum sensitization levels for rosetting and phagocytosis by U937 monocytes were 2,000 molecules IgG/cell for IgG3 and 5,000 molecules/cell for IgG1 mabs; maximum rosetting mediated by both IgG1 and IgG3 mabs was obtained at 15,000-20,000 molecules/cell. The IgG3 anti-D mabs were comparable to polyclonal anti-D in mediating binding of sensitized red cells to gamma-interferon stimulated monocyte-derived cultured macrophages and were markedly more effective than the IgG1 anti-D mabs. However, in lymphocyte ADCC assays, only anti-D mabs which were IgG1 Glm(3) were effective in mediating high levels of lysis of sensitized red cells, unlike the IgG1 Glm (1,17) or IgG3 G3m(21) mabs. Minimum sensitization levels required for this lymphocyte-mediated red cell lysis were found to be approximately 5,000 molecules/cell with one IgG1 Glm(3) mab; maximum lysis with this mab was obtained at 10,000 molecules/cell. Polyclonal anti-D containing both IgG1 and IgG3 was effective in all three assays. These observations suggest that different isotypes and allotypes of anti-D antibodies mediate red cell removed or destruction by monocyte or lymphocyte effector cell through functionally dissimilar Fc receptor interactions.     

IgG抗-E、抗-C引起配血不合1例

1病例资料患者,男,43岁,因尿毒症,肾性贫血急需血液透析入住我院,有输血史,贫血申请输注ABORh（D）同型悬浮红细胞2U,输血科在进行交叉配血时,盐水法相合、凝聚胺法主侧有凝集,次侧相合。经Rh血型系统鉴定,     

Flow-Cytometric Quantitation of Anti-D Antibodies

Objectives: Quantitation of Rh antibodies is important clinically in predicting the risk of hemolytic disease of the newborn. We describe a flow cytometry method for the quantitation of anti-D antibodies that we developed in parallel to a recently described method. Methods: As a secondary antibody we used whole IgG instead of F_ab molecules. The advantages, besides lower cost, include a stronge fluorescence signal with no need for amplification, and the possibility of diluting samples to minimize the risk of agglutination by IgM antibodies. We did extensive studies on reproducibility. Results: Reproducibility was superior to the autoanalyzer method. The two methods were roughly in agreement in estimating low, medium, or high levels of anti-D with a correlation coefficient of 0.89. The autoanalyzer measures the in vitro agglutination of all anti-D antibodies whereas flow cytometry measures the amount of IgG anti-D bound to red cells, which is more like the in vivo situation. Conclusion: Further studies in a clinical setting will show whether flow-cytometric quantitation may improve the diagnostic value of anti-D concentration measurement.     

Monoclonal antibodies in haematology

The development of methods for the production of monoclonal antibodies is having an important impact in the field of immunohaematology. Four separate areas are implicated. First, there is the use of monoclonal antibodies in blood transfusion, where antibodies within the ABO, Rh, Lewis, P, MN, Kell and Lutheran systems are available. Most of the monoclonal antibodies are of murine origin but the techniques for producing human monoclonal antibodies is now well established and this is especially valuable in the Rh system, with the production so far of anti-c, D, -E, -e and -G. Secondly, there is a great potential for the use of monoclonal anti-D to substitute for polyclonal anti-D in the prophylaxis of haemolytic disease of the newborn. The introduction of these antibodies will depend on clinical trials using both the IgG1 and IgG3 subclasses and on the ability to prepare antibody which is free of viruses and DNA. Thirdly, monoclonal antibodies are being used in basic research on red cell membranes to isolate and characterise blood group antigens. Finally, these antibodies are being used in bone marrow transplantation to purge the donor marrow of T-cells in order to reduce the incidence of graft-versus-host disease.     

The effect of naturally occurring Rh antibodies on the survival of serologically incompatible red cells

Investigations on six males with naturally occurring Rh antibodies are described. In two subjects in whom the antibody (one anti-E and one anti-D) could be detected only by a two-stage papain technique, the survival of incompatible red cells was normal. In the remaining four subjects, the antibodies (two anti-E and two anti-D) could be detected by the indirect antiglobulin test and, in these, incompatible red cells were destroyed at an accelerated rate; in two of the subjects, 75-99% of the cells were cleared within 24 h; in the other two, 50% of the cells were cleared within 24 h and the remaining cells were cleared far more slowly. All six antibodies were mainly or wholly IgG; a clear-cut immune response was observed in only one case.     

Heterogeneity of IgG1 monoclonal anti-Rh(D): an investigation using ADCC and macrophage binding assays

Three monoclonal IgG1 anti-Rh(D), UCH D4, ARC 7D5 and UKTS FC3, produced by Epstein-Barr virus transformed cells from Rh(D)-sensitized individuals, were compared with polyclonal single donor anti-D sera and therapeutic immunoglobulin preparations in antibody dependent cellular cytotoxicity (ADCC) and macrophage binding tests. When assayed at equal anti-D concentrations monoclonal antibodies varied considerably in their ADCC and macrophage binding activities: only UKTS FC3 showed significant activity in both assays, but these were substantially lower than those of the polyclonal anti-D sera and immunoglobulins. When examined in different combinations the monoclonal antibodies showed little synergism in mediating red cell destruction by the effector cells. Factors which might contribute to the diverse ADCC and macrophage binding activities of the monoclonal anti-Ds of the same IgG subclass are discussed.     

Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers

Prophylactic anti-D is a very safe and effective therapy for the suppression of D-immunization and prevention of haemolytic disease of the foetus and newborn. The primary mode of action of anti-D is rapid clearance of fetal D-positive red cells from the maternal circulation, mediated by interactions with immunoglobulin G Fc receptors on macrophages in the spleen. Many anti-D monoclonal antibodies (mAb) have been produced by a variety of methods. Twelve anti-D mAbs were tested in eight studies for their ability to mediate clearance of autologous red cells, and 13 antibodies studied in seven trials of the clearance of D-positive red cells injected into D-negative subjects. Antibodies produced by human B-cell lines, mouse-human heterohybridomas and Chinese hamster ovary cells varied in their activity with none being quite as effective as polyclonal anti-D. However, clearance mediated by recombinant anti-D produced by rat YB2/0 cells was extremely rapid, faster than polyclonal anti-D, but with haemolysis and some hepatic accumulation of red cells observed in one study. Two human anti-D mAbs prevented D-immunization. In contrast, anti-D mAbs from heterohybridomas increased the incidence and rapidity of anti-D responses. It is hypothesised that unnatural glycosylation of monoclonal anti-D produced by some cell lines may have caused these unexpected results. In some antibodies, unusual oligosaccharides on anti-D may have affected binding to Fc receptors resulting in reduced red cell clearance. For others, non-human glycoforms of anti-D might have bound to innate immune recognition molecules promoting pro-inflammatory reactions. These extensive data on the clinical activity of monoclonal anti-D produced by cell lines derived from four species will inform the future development of monoclonal anti-D for RhD prophylaxis.     

Providing saline reacting anti-D cell typing reagent.

Since 1969, when anti-D specific IgG was made available to protect recently delivered D negative women from sensitisation by fetal D positive cells, the supply of saline acting anti-D, suitable for reagent use has diminished. The same immunized male blood donors that provide the anti-D for the production of the specific IgG used for prophylaxis may, when bled up to fourteen days after restimulation, provide a source of saline anti-D suitable for reagent use. The results of such a procedure in 12 donors are described.     

Characteristics of Different Rhesus Alloantibodies in Lymphocyte Dependent (K Cell) Cytotoxicity to Human Erythrocytes

The property of rhesus alloantibodies to elicit antibody-dependent, cell-mediated cytotoxicity (ADCC) against target erythrocytes carrying various Rh genotypes was studied. The killer activity of normal peripheral lymphocytes on human erythrocyte target cells carrying the appropriate antigens elicited by alloantisera was measured by 51Cr release at 18 h. There was no correlation between ADCC and antibodies directed to the antigens present on the surface of different genotypes of Rh-positive red blood cells. The agglutinin titre of different Rh antibodies showed no correlation with the level of ADCC although the degree of cellular cytotoxicity was different with different anti-D sera. Anti-C + D+ E antibody caused higher ADCC than anti-C + D and the lowest cytotoxicity was observed with anti-D and anti-D+ E. This raised the possibility that ADCC was elicited by antibodies directed to other specificities. K cell lysis of human red cells by human peripheral blood lymphocytes in vitro suggests that a similar mechanism may operate in vitro in the destruction of erythrocytes coated by allo or autoantibodies.     

Providing saline reacting anti-D cell typing reagent

Summary Since 1969, when anti-D specific IgG was made available to protect recently delivered D negative women from sensitisation by fetal D positive cells, the supply of saline acting anti-D, suitable for reagent use has diminished. The same immunized male blood donors that provide the anti-D for the production of the specific IgG used for prophylaxis may, when bled up to fourteen days after restimulation, provide a source of saline anti-D suitable for reagent use. The results of such a procedure in 12 donors are described.