anti-D mAbs Research Articles

Development of acceptance criteria for the results of anti-D IgG quantification in human anti-D (Rh<sub>о</sub>) immunoglobulin preparations by enzyme immunoassay

The competitive enzyme-linked immunosorbent assay (ELISA) used for the quantification of specific antibodies, anti-D (Rho) IgGs, in human anti-D (Rho) immunoglobulin preparations relies upon the competition between anti-D antibodies in the preparation and anti-D monoclonal antibodies (mAbs) for immunochemical binding to D-antigen epitopes of the immunosorbent, human erythrocytes of the ccDEE phenotype immobilised on a solid support. The immunosorbent is prepared in-house under laboratory conditions. The certification of the International Standard (IS) for anti-D immunoglobulin included attempts to use the CCDee phenotype, which is more common (16.01%) than the ccDEE phenotype (2.16%). It is possible to use the CCDee phenotype, as long as the user adheres to the acceptance criteria for the results, developed during method validation. The aim of this study was to develop the acceptance criteria for the results of anti-D IgG quantification in human anti-D immunoglobulin preparations by the ELISA method that would allow using CCDee erythrocytes to prepare the immunosorbent should there be no erythrocytes of the ccDEE phenotype available. Materials and methods: the study used human anti-D immunoglobulin preparations; anti-D IgG–spiked samples; the IS for anti-D immunoglobulin; the reference standard (RS) for anti-D IgG–free immunoglobulin; anti-D mAbs; and erythrocytes of the CCDee, ccDEE, and ccddee phenotypes. The authors quantified anti-D IgG antibodies by the competitive ELISA. The statistical analysis used parametric and nonpar ametric tests. Results: the study demonstrated the possibility of using CCDee erythrocytes for competitive immunochemical binding with anti-D mAbs and anti-D IgG of various human anti-D immunoglobulin preparations. The suitability of the analytical procedure with the CCDee phenotype was confirmed by validation parameters: trueness, intermediate precision, linearity, selectivity, specificity, and robustness of the method and comparability of the results obtained when using the CCDee and ccDEE phenotypes. The authors developed the acceptance criteria: the relative values of anti-D IgG content in the test sample should range within ±20% of the nominal value; the coefficient of determination (R2) should be at least 0.9; the relative standard deviation (RSD, %) of three absorbance values for each concentration should not exceed 20%. Conclusions: these acceptance criteria guarantee the reliability of assay results of anti-D IgG quantification by the ELISA, which allows them to be used by specialists of control and analytical laboratories to assess the quality of human anti-D immunoglobulin preparations.

Abstract A01: Ex vivo evaluation of an anti-Müllerian hormone type II receptor humanized antibody with optimized Fc effector function in ovarian cancer

Abstract Purpose of the study: Epithelial ovarian cancer (OC) is one of the most common gynecologic malignancies and the fifth most frequent cause of cancer death in women. Paclitaxel and Carboplatin chemotherapy (CT) remains the standard treatment, but tumor relapses in approximately 80% of patients, mostly due to drug resistance. Novel therapeutic options are therefore strongly required. Recently, human Anti-Müllerian Hormone type II Receptor (AMHRII) has been identified as a potential target, but up to date the AMHRII expression in ovarian tumors has been poorly documented. A humanized glyco-engineered monoclonal antibody (mAb) called 3C23K with higher Fc effector function has been developed. The aim of this study is to investigate the potential efficacy of this mAb by assessing AMHRII expression and analyzing the localization and amount of mAb responsive immune effectors in OC patient and in a patient-derived xenograft (PDX) model. Experimental procedures: AMHRII expression was prospectively studied in fresh biopsies (primary ovarian tumors, peritoneal carcinomatosis and ascites) from a cohort of 28 patients, by means of both flow cytometry (FC) and immunofluorescence (IF). We have characterized the intratumoral immune infiltrate by FC and determined the localization of immune cells within the tumor by IF. Similar experiments were performed on an undifferentiated highly proliferative serous ovarian adenocarcinoma PDX model, in parallel to in vivo efficacy study of 3C23K alone or in combination with standard CT. The antitumor activity of infiltrating immune cells isolated from OC patients (peritoneal washing) was assessed using red blood cells as target and a glyco-engineered anti-D Rhesus mAb. The antibody-dependent cell cytotoxicity (ADCC) and the antibody-dependent cell phagocytosis (ADCP) abilities of 3C23K against AMHRII+ cells (COV434-RII) were analyzed by IF using monocyte-derived macrophages (MDM) from blood of healthy donors as effector cells. Summary of the data: Here, we show that among the 28 ovarian tumors tested, 19 patients (68%) were positive for AMHRII with at least 15% of tumor cells being positive for AMHRII. OC are largely composed of (CD45+) immune cells mostly composed of tumor associated macrophages (TAMs) and tumor infiltrating lymphocytes (TILs). TAMs highly express M2-like markers, such as CD206, but also strongly express the Fcγ III receptor (CD16). Unlike TILs, which accumulate in the peritumoral stroma, CD16+ TAMs are found in both the stroma and the tumor islets. Moreover, tumors infiltrating immune cells are able to induce a mAb-dependent cytotoxicity ex vivo. In parallel, in vivo experiments have shown an intermediate efficacy of the 3C23K antibody administered alone and a significantly increased antitumor effect in combination with CT including a prolonged survival. IF and FC analyses showed high TAM infiltration compatible with induction of ADCP by 3C23K in this model. Furthermore, 3C23K mAb induces ADCC and/or ADCP of COV434-RII cells by MDM likely through CD16 interaction, either in the absence or presence of saturating concentrations of Ig. Conclusions: We have confirmed the expression of AMHRII in 2/3 of OC patients. MAb responsive immune effectors and particularly macrophages largely infiltrate OC and are found in contact with malignant cells. Targeting AMHRII by the 3C23K mAb dramatically increases the efficacy of a standard CT in vivo. 3C23K appears to act through macrophage-induced ADCC and/or ADCP of tumor cells and current studies are designed to refine its mechanism of action. Our results indicate that most ovarian tumors display optimal conditions for a 3C23K-based immunotherapy. Hence, the combination of 3C23K with CT could bring additional therapeutic benefit in OC patients compared to standard chemotherapy. Citation Format: Houcine Bougherara, Fariba Némati, Christophe De Romeuf, Jean-Marc Barret, Gérald Massonnet, Marie-Aude Le Frère-Belda, Jean-François Prost, Didier Decaudin, Emmanuel Donnadieu. Ex vivo evaluation of an anti-Müllerian hormone type II receptor humanized antibody with optimized Fc effector function in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A01.

Do YB2/0 cells produce alien sugars?

Olovnikova et al. ("Impact on N-glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties" (2012) Biochemistry (Moscow), 77, 925-933) mentioned the presence of "alien sugars" on monoclonal antibodies (mAbs) produced by YB2/0 cell line. We summarize in this paper our previous findings on the glycosylation profile of two anti-D mAbs produced in this cell line (LFB-R297 and LFB-R593, so-called Roledumab). Our results show the absence of any immunogenic glycotopes, and furthermore neither immunogenicity nor other serious adverse reactions were observed during clinical trials.

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for suppressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human transformed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.

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.

Section 1A: Rh serologyCoordinator’s report

One hundred forty-two Rh-specific monoclonal antibodies (Mabs) were evaluated by serology in 27 laboratories. Evaluators were asked to test each Mab at three dilutions in specified serological techniques against normal positive and normal negative phenotype cells, and any Rh variant cells that they had available. Raw data was submitted to the coordinator for overall analysis. Results were analysed by expressing the sum of reaction grades for each Mab with each variant cell as a percentage of the sum of reaction grades of that Mab with normal phenotype cells. Anti-D Mabs were sorted into 23 groups which had the same pattern of reactions with different partial D phenotype cells. Eighteen of these corresponded to previously defined patterns; five were new patterns. Combined with data from the previous workshop, this means that 30 different reaction patterns have been defined. A new nomenclature is introduced for numbering the epitopes. Reactions with new variants DNB, DNU and DAR indicated some further subsplits of these patterns. Reactions with Category Va cells indicated that there were five different types of Va cells that could be distinguished serologically with monoclonal antibodies. No patterns of reactivity corresponding to the epitope groups could be observed with the different types of weak D tested. Anti-E Mabs were sorted into 14 groups, and the E variant cells into seven groups.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator’s report

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.

Coordinator's report: an assessment of the functional activity of human Rh monoclonal antibodies after their evaluation by nine laboratories.

The functional activity of 55 anti-D and 26 MAbs of other Rh specificities was determined as part of the Third International Workshop and Symposium on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens. Most MAbs were IgG1 (45 anti-D, 16 Rh). There was a large range of anti-D and IgG concentrations of the anti-D MAbs (0.4-1680 IU/ml, 1.5-400 micrograms/ml). Correlation between quantitative data from different laboratories was good. Six laboratories tested the anti-Ds in monocyte ADCC assays. Methods varied greatly, especially the effector cells used, the methods of red cell sensitisation, the effector to target cell ratio and the assay incubation times. There was some correlation of results between most laboratories, but results were better when similar assays were performed. The extent of monocyte-mediated haemolysis was related to the number of molecules of IgG anti-D bound to the red cells. Monocyte phagocytosis assays resulted in a greater variability in results between the four evaluating laboratories, though IgG3 MAbs were found more active than IgG1, and mostly promoted red cell adherence rather than phagocytosis. Two monocyte chemiluminescence assays found comparatively low activity of the IgG3 MAbs; the most active IgG1 anti-Ds were MAbs 93 and 95. Correlation between different monocyte-mediated assays was generally poor unless the assays were performed by the same laboratory. Results of a macrophage ADCC assay showed that the IgG3 anti-D's promoted greater haemolysis than most IgG1 MAbs. Only two IgG1 anti-D MAbs (70 and 104) mediated high haemolysis in this assay. Lymphocyte ADCC assays were utilised in four laboratories. The IgG3 antibodies exhibited low activity but one IgG1 anti-D (104) consistently mediated high haemolysis. There was no relationship between quantitation and haemolysis in this assay. A few MAbs exhibited low or no activity in most assays, mainly due to low quantitation. Most of the MAbs of non-D specificity mediated low functional activity which in most cases was not due to low quantitation. However two MAbs, 24 (anti-CcEe) and 25 (anti-CD), consistently showed good activity. This study highlighted the great variability in assay methodology between the nine participating laboratories, which resulted in some apparent differences in functional activity of some of the MAbs. The use of standardised methods as well as fewer MAbs tested and quantitated at different concentrations may help to determine the relevant factors contributing to IgG function.

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.

IgG anti-D MAbs in tests by flow cytometry.

Seventy-two IgG anti-D Mabs were studied by indirect immunofluorescence and flow cytometry (FCM) with R1r, rr and various D variant phenotype red blood cells (RBCs) (DIIIb,DVI,DVII,Rh33 and uncategorized variant ISBT number 48). The results were compared with those obtained by indirect antiglobulin test (IAT).

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.

Demonstration of autoreactivity by a human monoclonal IgG anti‐Rh D antibody

Human IgG monoclonal antibodies (mabs) against the Rh D antigen have considerable potential for the prophylaxis of haemolytic disease of the newborn. We have carried out in vitro testing for cross-reactions with tissue components by screening two such mabs against animal tissues and a wide panel of human organs from nearly 50 individuals, most of whom were of known Rh D phenotype. Cryostat sections were studied by indirect immunohistochemical techniques. One of the mabs showed non-specific, dose-dependent binding to multiple tissue components whereas the other specifically and consistently reacted strongly with animal smooth muscle and human smooth muscle (vascular, in the walls of hollow viscera, in the respiratory tract) from both Rh D-positive and -negative donors. Immunoprecipitation experiments identified the probable smooth muscle antigen as actin or actin-associated. However, on the basis of inhibition experiments, and by direct estimation of the association constant, the affinity of this mab for smooth muscle was lower than that for Rh D. These results demonstrate autoreactivity by an IgG anti-D mab and show differences in the immunochemical characteristics of human anti-D mabs which may be clinically relevant.

Diversity of human anti-D monoclonal antibodies revealed by reactions with chimpanzee red blood cells.

Fifty-three human anti-D monoclonal antibodies (mAbs) revealed a striking diversity of reactions in tests with panels of chimpanzee red blood cells (RBCs) of various R-C-E-F blood group phenotypes (counterparts of the human Rh-Hr groups). The reactivities of these antibodies, which depended on the agglutination technique used, could be classified into four main types. These patterns of reactivity of anti-D mAbs with chimpanzee RBCs showed only limited correlation with types of reactions observed with human D variant RBCs. Primate red cells may, therefore, constitute an independent test system for subclassification of human monoclonal antibodies. Comparison of reactivities of human anti-D mAbs with chimpanzee and human D variant RBCs confirms the homology between the chimpanzee Rc, and the human D antigens. The chimpanzee Rc shares with human D the epitopes epD5, epD6/7 and epD8, but lacks epitopes epD1, epD2, epD3 and epD4 of the Rh mosaic, thus resembling the human D variants IVb and Vc.

Haemolysis mediated by anti-D monoclonal antibodies in direct and cold target competition ADCC assays.

Thirteen IgG anti-D human monoclonal antibodies (mAbs) were compared for their ability to mediate lysis of D-positive erythrocytes by PBMC in direct and cold target competition antibody-dependent cell-mediated cytotoxicity (ADCC) assays. In the latter assay, lysis of fluid-phase anti-D-sensitised O Rh D-positive papainised erythrocytes (E-IgG) was inhibited by A (or B) Rh D-negative papainised erythrocytes sensitised by fluid-phase anti-A (or anti-B) mAbs. The competitive and lytic activities of the anti-D mAbs were characterised by the equilibrium dilution (ED) values, which were the reciprocal of the dilution of anti-A (or anti-B) at which lysis of target E-IgG and competitor E-IgG were identical. There was a poor correlation between the number of erythrocyte-bound anti-D molecules and either the sensitivity of E-IgG anti-D to haemolysis in the direct ADCC assay, or to the ED values of the mAbs obtained in the cold target competition ADCC. The discriminatory power of the cold target competition ADCC was better than than of the direct ADCC to detect differences in the lytic potential of the anti-D mAbs.

Diversity of Human Anti-D Monoclonal Antibodies Revealed by Reactions with Chimpanzee Red Blood Cells

Fifty-three human anti-D monoclonal antibodies (mAbs) revealed a striking diversity of reactions in tests with panels of chimpanzee red blood cells (RBCs) of various R-C-E-F blood group phenotypes (counterparts of the human Rh- Hr groups). The reactivities of these antibodies, which depended on the agglutination technique used, could be classified into four main types. These patterns of reactivity of anti-D mAbs with chimpanzee RBCs showed only limited correlation with types of reactions observed with human D variant RBCs. Primate red cells may, therefore, constitute an independent test system for subclassification of human monoclonal antibodies. Comparison of reactivities of human anti-D mAbs with chimpanzee and human D variant RBCs confirms the homology between the chimpanzee Rc, and the human D antigens. The chimpanzee Rc shares with human D the epitopes epD5, epD6/7 and epD8, but lacks epitopes epD1, epD2, epD3 and epD4 of the Rh mosaic, thus resembling the human D variants IVb and Vc.

FUNCTIONAL ACTIVITY OF HUMAN Rh MONOCLONAL ANTIBODIES (MABS)

Anti-D and anti-c monoclonal antibodies (MABS) were found to be heterogeneous with respect to their activity in five cell-mediated functional assays. The two IgG3 MABS promoted greater rosette formation of red cells with monocytes than the IgG1 MABS. Discrepant results were obtained by different laboratories for the relative effectiveness of the MABS in promoting monocyte-mediated red cell phagocytosis and lysis, which may have been due to variations in the assay methods used. The IgG3 anti-D MAB promoted greater monocyte chemiluminescence than the IgG3 anti-c MAB, but of these two MABs only the anti-c mediated lysis of red cells in lymphocyte ADCC assays. The majority of the IgG1 anti-D MABS were ineffective at promoting red cell lysis by lymphocytes (K cells), but two were active even when diluted 1 in 300. There was no correlation of functional activity with red cell sensitization levels, IgG concentration or D epitope specificity of the MABS.

Heterogeneity in the ability of IgG1 monoclonal anti‐D to promote lymphocyte‐mediated red cell lysis

Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.

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.