Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.

Ultrastructural localization of glycoprotein IIIa (GPIIIa, beta 3 integrin) on placental syncytiotrophoblast microvilli: implications for platelet alloimmunization during pregnancy.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a more commonly occurs in first pregnancies, unlike hemolytic disease of the newborn. Anti-D is produced after D+ fetomaternal hemorrhage; this usually occurs at parturition. Anti-HPA-1a could develop during pregnancy if maternal immunization is stimulated by HPA-1a expressed not only on platelets but also on other fetal cells. STUDY DESIGN AND METHODS: An ultrastructural study of fetal placental chorionic villi was undertaken to determine the localization of glycoprotein (GP)IIIa carrying the HPA-1a/1b polymorphism. First trimester and term villi were incubated with a monoclonal antibody (MoAb) to GPIIIa or with positive control MoAbs (anti-placental alkaline phosphatase and ED822 MoAb) to villous syncytiotrophoblast (ST). Binding of MoAbs was detected with a gold-conjugated secondary antibody before processing the tissues and examination of ultrathin sections in an electron microscope. RESULTS: Gold particles were evident on microvilli on the apical surface of ST when labeled with anti-GPIIIa and the placenta-specific MoAbs but not with an isotype control antibody. Immunolabeling for anti-GPIIIa on first trimester ST was similar to that of term ST. CONCLUSION: The apical surface of the ST is bathed in maternal blood. During the natural regenerative process of human placenta, senescent parts of the ST are shed into maternal blood during pregnancy. This includes both apoptotic ST nuclei and microparticulate ST debris. The presence of GPIIIa on this circulating ST cellular material could be the source of HPA-1a alloantigen causing primary immunization of susceptible primigravidae early enough for anti-HPA-1a to cause fetal thrombocytopenia during a first pregnancy.

Blood doping in athletes--detection of allogeneic blood transfusions by flow cytofluorometry.

Athletes may undergo blood transfusion to increase their red cell mass and the oxygen carrying capacity of their blood in order to confer a competitive advantage. Allogeneic transfusions are normally mismatched at one or more minor blood group antigens. The most sensitive and accurate method known to detect this form of blood doping is flow cytometry. Low percentages of antigen-positive and antigen-negative red blood cells (RBCs) can be quantitated using suitable specific alloantibodies and careful analysis. By testing blood samples taken at various times, a reduction in the percentage of a minor population of RBCs will indicate transfusion has occurred.

Monoclonal anti-D development programme.

Administration of anti-D immunoglobulin to D- women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn. The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D. Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (Fc gammaR) were selected for clinical studies. They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors. The mean half-lives of BRAD-3 and BRAD-5 in D- subjects were 10.2 and 22.2 days, respectively. The clearance of D+ red cells injected into D- subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5/BRAD-3). The clearance rate was related to the amount of anti-D on the red cells. Clearance of the D+ red cells coated with BRAD-3 was more rapid in subjects homozygous for Fc gammaRIIIa-F/F158 than in those expressing the Fc gammaRIIIa-V158 allele. The subjects were protected from Rh D immunisation. A large multi-centre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D- subjects given 400 microg i.m. 24 h after injection of 5 ml D+ red cells. Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses. There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28. Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis. The responder rate was 13% by week 36 and 24% by week 48. There was no relationship between HLA haplotype and Rh D immunisation. The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects. The most likely mechanism of action was considered to be inhibition of B cells resulting from co-cross-linking antigen receptors with inhibitory Fc gammaR when the B cells contacted red cells that had bound passive anti-D.

On the mechanism of tolerance to the Rh D antigen mediated by passive anti-D (Rh D prophylaxis).

Anti-D prophylaxis is the most successful clinical application of antibody-mediated immune suppression. Passive IgG anti-D is given to Rh D-negative women to prevent immunisation to foetal Rh D-positive red blood cells (RBC) and subsequent haemolytic disease of the newborn. Despite its widespread use and efficacy, the mechanism of action of this therapy is unproven. The known facts about the antigen, antibody response, dose of anti-D, RBC clearance and effects of the passive anti-D on subsequent primary and secondary immune responses are discussed in relation to recent information on ways by which immune responses may be suppressed. Most Rh D antigen sites on RBC are not bound by passive anti-D, and thus epitope masking (which may occur in experimental murine models using xenogeneic RBC) is not the reason why anti-D responses are prevented by administration of prophylactic anti-D. It is hypothesised that although clearance and destruction of the antigenic RBC may be a contributing factor in preventing immunisation, down-regulation of antigen-specific B cells through co-ligation of B cell receptors and inhibitory IgG Fc receptors must also occur.

In vivo studies of monoclonal anti-D and the mechanism of immune suppression.

Administration of anti-D immunoglobulin to D- women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn. The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D. Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (Fc gamma R) were selected for clinical studies. They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors. The clearance of D+ red cells injected into D- subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5: BRAD-3). The subjects were protected from Rh D immunisation. A large multicentre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D- subjects given 400 micrograms i.m. 24 hours after injection of 5 ml D+ red cells. Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses. There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28. Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis. The responder rate was 13% by week 36 and 24% by week 48. The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects. The most likely mechanism of action was considered to be inhibition of B cells resulting from co-crosslinking antigen receptors with inhibitory Fc gamma R when the B cells contacted red cells that had bound passive anti-D.