Human Monoclonal anti-D Antibodies Research Articles

Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets

AbstractRh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis.

The Specificity and Isotype of Monoclonal Anti-D Antibodies Dictates Their Ability To Inhibit Opsonized Platelet Phagocytosis.

The use of polyclonal anti-D is the established treatment for prophylaxis against rhesus immunization in Rh(D) negative pregnant women and is effective in raising the platelet counts in patients with autoimmune thrombocytopenic purpura (AITP). Since the supply of plasma derived anti-D antibodies is limited and plasma products carry the risk of infectious complications, replacement with monoclonal anti-D antibodies is a promising alternative. This study analyzed the efficacy of polyclonal anti-D (WinRho®SDF, Cangene) and six human monoclonal anti-D antibodies with differeing isotypes and specificities (Table 1) for their ability to opsonized erythrocytes and modulate monocyte phagocytosis of opsonized platelets in a flow cytometric assay. Human platelets were labeled with CellTracker CMGreen, opsonized with various HLA or HPA1a specific antibodies, incubated with the monocytic leukemic cell line THP-1 for 2 h and intracellular fluorescence was compared at 4°C and 37°C. Various concentrations of anti-D opsonized erythrocytes were added to the assay and the changes in intracellular fluorescence was compared. The results demonstrate that the THP cells significantly phagocytosed opsonized platelets in an Fc-dependent manner e.g. the fold change in median channel fluorescence between 37°C and 4°C for intact IgG W6/32 opsonized platelets was 51.5 ± 5.2 (mean+SD, n=74) and only 2.1+2.1 if the platelets were labeled with F(ab')2 fragments. When erythrocytes were opsonized with the monoclonal anti-D antibodies that shared specificity but differed in IgG isotype (RhG1/RhG3 and BRAD3/BRAD5) and added to the assay, the IgG3 isotypes had a significantly greater ability to inhibit opsonized platelet phagocytosis compared with IgG1 isotypes and this inhibition was similar to that observed with polyclonal anti-D opsonized erythrocytes. However, in the one case where the monoclonal anti-D antibodies shared isotypes but differed in specificity (113/178), rRh9B8-113 monoclonal antibody significantly inhibited platelet phagocytosis whereas the rRh429-178 antibody did not. These results suggest that some human monoclonal antibodies can be as efficient as polyclonal anti-D in inhibiting opsonized platelet phagocytosis and the degree inhibition depends on the IgG isotype and specificity of the monoclonal antibodies. Thus, a defined cocktail of monoclonal anti-D antibodies could be prepared to mimic the effects and replace plasma-derived polyclonal anti-D.Table 1:Characteristic of the monoclonal anti-D antibodies.AntibodyIsotypeSpecificityrRh9B8-113IgG1Loop 3rRh429-178IgG1Loop 4RhG1IgG1Loop 6RhG3IgG3Loop 6BRAD3IgG3Loop 6BRAD5IgG1Loop 6

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.

Comparative study of human monoclonal anti-D antibodies of IgG and IgM classes in tests with red cells of nonhuman primates

Forty-nine human anti-D (Rho) monoclonal antibodies of the IgG and IgM classes were tested with red blood cells of various nonhuman primates, from anthropoid apes to Prosimians, and significant differences in reactivity were observed among antibodies of two classes depending on taxonomic position of primate animals. By and large, higher percentage of IgM mAbs gave positive reactions with nonhuman primate red cells and, particularly, with blood of lower monkeys: Old and New Worlds monkeys and Prosimians, than did those of IgG class. Allotypic reactions with red cells of African apes were produced by majority of IgG mAbs but by very few IgM reagents. Some of those reactions defined epitopes related to human D, such as chimpanzee Rc and gorilla Dgor. By contrast, individual differences among Old World monkey species were revealed almost exclusively in tests using anti-D mAbs of IgM class. Some of the epitopes detected by these antibodies on the red cells of macaques are related to human D alloantigen, as confirmed by absorption experiments. Differences among mAbs evidenced in tests with nonhuman primate red cells reflect the complexity of the immune reactions to the human D antigen.

The R‐C‐E‐F blood group system of chimpanzees: Serology and genetics

Six chimpanzee alloimmune antibodies define 20 phenotypes of the R-C-E-F blood group system, the counterpart of the human Rh system. Of the several specificities of this system, the Rc constitutes the crucial link with human Rh since the reactions of some chimpanzee alloimmune anti-Rc sera with human red cells parallel those obtained with human anti-Rho reagents. Reciprocally, properly absorbed human anti-Rho sera detect Rc specificity on chimpanzee red cells. Tests with large panels of human monoclonal anti-D antibodies confirm the notion of shared epitopes between human alloantigen Rho(D) and chimpanzee alloantigen Rc.

Differential exposure of epitopes on the human red cell antigen D (Rh)

The D polypeptide of the human Rh blood group system has a number of different epitopes on its surface. It is also known that there is considerable variation in the number of D antigen sites available to different human monoclonal anti-D antibodies. For instance, certain monoclonal antibodies recognise only a small number of sites on the red cell surface (about 9,000 sites/red cell on R1R2 cells) whereas other antibodies recognise a high number (about 20,000-30,000 sites/red cell). It has been found that cholesterol enrichment of the red cell membrane increases the number of sites available to those antibodies which recognize only a few sites but has no effect on those recognising many sites. The results are consistent with the view that access to some of the D epitopes is partially hindered by neighbouring molecules in the membrane and that alteration of the lipid content of the membrane changes it in such a way as to allow increased access to these obstructed epitopes.

Multiple Epitopes Recognised by Human Monoclonal IgM Anti‐D Antibodies

Bivalent 7S subunits were obtained from 7 purified IgM human monoclonal anti-D antibodies with 33-63% of the total protein-retaining functional binding activity. The number of sites per red cell recognized by these subunits ranged between 9,400 and 28,500. The values are taken to indicate that a number of different epitopes on the D polypeptide are being recognized. There was competition between IgG and IgM antibodies for binding to the red cells, indicating that both classes of antibody are recognizing epitopes on the same D polypeptide. The value of the functional affinity constants for the binding of the 7S subunits varied between 1.0 and 8.8 x 10(7) M-1 and there was a 3- to 16-fold increase on reduction of the ionic strength (0.05 M NaCl). Using agglutination in microwells, the end-point of the titres for the native pentamer IgM antibodies occurred at concentrations in the range 7-26 ng/ml, with one exception where the concentration required was 165 ng/ml.

Multiple Epitopes Recognised by Human Monoclonal IgM Anti-D Antibodies

Bivalent 7S subunits were obtained from 7 purified IgM human monoclonal anti-D antibodies with 33-63% of the total protein-retaining functional binding activity. The number of sites per red cell recognized by these subunits ranged between 9,400 and 28,500. The values are taken to indicate that a number of different epitopes on the D polypeptide are being recognized. There was competition between IgG and IgM antibodies for binding to the red cells, indicating that both classes of antibody are recognizing epitopes on the same D polypeptide. The value of the functional affinity constants for the binding of the 7S subunits varied between 1.0 and 8.8 x 10^7 M^-1 and there was a 3- to 16-fold increase on reduction of the ionic strength (0.05 MNaCI). Using agglutination in microwells, the end-point of the titres for the native pentamer IgM antibodies occurred at concentrations in the range 7-26ng/ml, with one exception where the concentration required was 165 ng/ml.

Detection of Rh D‐associated epitopes in human and animal tissues using human monoclonal anti‐D antibodies

The distribution of Rh D-associated epitopes in human and animal tissues has been assessed by immunochemical techniques using six human monoclonal anti-D antibodies. The IgM antibody MAD-2 reacted predominantly with endothelial cells lining blood vessels and with animal leucocytes and human leucocytes from both Rh D-positive and Rh D-negative donors. Immunoblotting revealed reactivity with a 55 kDa tissue component which was most prominent in homogenates of heart, lung and spleen. In contrast, the IgG antibodies UCHD4 and FOG-1 stained smooth muscle, and UCHD4 in addition weakly stained cardiac and skeletal muscle and the glomeruli of kidney. Immunoblotting with UCHD4 revealed reactivity with a 27 kDa tissue component which was most prominent in heart homogenates. The results show that the six monoclonal anti-D antibodies recognize at least four different epitopes and that Rh D-associated epitopes may occur in non-erythroid cell types.

Demonstration of Seven Epitopes on the Rh Antigen D Using Human Monoclonal Anti-D Antibodies and Red Cells from D Categories

The agglutination patterns have been established for the reaction between 29 monoclonal antibodies with specificity for the Rh antigen D and red cells of D categories IIIa, IIIc, IVa, IVb, Va, Vc, VI and VII, which are known to lack certain epitopes on the D polypeptide. Six different agglutination patterns were recognized and interpreted to indicate the recognition of seven different epitopes. These epitopes are termed epD1 through to epD7. The separate existence of epD6 and epD7 is deduced from previous observations in inhibition studies using purified 125 I-labelled antibodies; they cannot yet be distinguished in agglutination tests. The number of epitopes lacking from cells of each category varied between two and five. As all the antibodies agglutinated cells of categories IIIa, IIIc and VII and cells of categories II, IIIb and Vb were not available, it is probable that there are epitopes other than the seven presently recognized. Eighteen out of the 29 antibodies which were examined recognized epitopes epD6/7 and it is suggested either that antibodies recognizing these epitopes predominate in polyclonal anti-D sera, or that the lymphocytes producing these antibodies are preferentially selected during establishment of cell lines.

Immunogenic and Antigenic Epitopes of Immunoglobulins Binding of Human Monoclonal Anti-D Antibodies to FcRI on the Monocyte-Like U937 Cell Line

Immunogenic and Antigenic Epitopes of Immunoglobulins Binding of Human Monoclonal Anti-D Antibodies to FcRI on the Monocyte-Like U937 Cell Line