Is an expression system for producing therapeutic antibodies with immunosuppressive properties found at last? Comment to letter by Dr. Quagliaroli.

The prophylaxis of the hemolytic disease of the newborn - a mandatory procedure in obstetrics - requires significant amounts of plasma-derived polyclonal anti-D immunoglobulin. Despite numerous attempts, the proper technology for mass production of effective monoclonal anti-D is still not available. LFB Biotechnologies is currently performing clinical trials with recombinant anti-D antibody that has low fucose content and is expressed in the cells of rat myeloma YB2/0. It was shown that this drug is well tolerated, accelerates fast clearance of D+ red blood cells, and can inhibit anti-D immune response in Rhesus-negative volunteers.

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.

Effector properties and glycosylation patterns of recombinant human anti-D-IgG1 antibodies produced by human PER.C6(®) cells.

Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

[Immunoglobulin G monoclonal human anti-rhesus Rho(D) to prevent rhesus-incompatibility]. / Immunoglobulin G monoklonal'nyi chelovecheskii anti-rezus Rho(D) dlia profilaktiki rezus-nesovmestimosti.

RhD immunisation which follows pregnancy can be prevented by administration to the rhesus-negative mother of 20 micrograms anti-D immunoglobulin per 1 ml of D-positive fetal red cells in the maternal circulation. With the aim of substitution of polyclonal anti-D with monoclonal one we developed human-mouse cell lines producing anti-RhD IgG1 by fusing EBV-transformed human immune lymphocytes with murine myeloma. After several clonings and passages the EBV genome was eliminated from the cell lines. The antibodies were purified from culture supernatants using protein A affinity chromatography and tested for sterility, virus contamination, pyrogenecity, toxicity and DNA content. The monoclonals were compared with the standard polyclonal anti-RhD in in vitro and in vivo assays. In antibody-dependent cell cytotoxicity (ADCC) 2 of 4 studied monoclonals promoted greater RBS lysis than polyclonal anti-D at equivalent concentrations. Detection of binding site number of monoclonals anti-D revealed about 10,000/RBS D-determinants on DCe/dce erythrocyte which agrees with the data for polyclonal anti-D. Best in ADCC monoclonal anti-D sharply increased human autologous 51Cr-labelled rbc sensitised in vitro as well as accelerated clearance of RhD RBS from circulation of Rh-negative volunteers injected with 150 micrograms monoclonal anti-D. After clinical trial using of monoclonal anti-D is permitted in Russia.

[Monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn]. / Monoklonal'nyi anti-D immunoglobulin dlia profilaktiki gemoliticheskoi bolezni novorozhdennykh.

Stable human-mouse heterohybridoma producing human monoclonal anti-D IgG1 has been derived by fusing Epstein-Barr virus (EBV) transformed human immune lymphocytes with mouse myeloma. The absence of EBV DNA in heterohybridoma was demonstrated by the polymerase chain reaction. The antibody was purified by affinity chromatography on protein A Sepharose and tested for sterility, pyrogenicity and toxicity according to the Pharmacopoeia XI and the ability to clear from circulation RhoD-positive red cells. The time of clearance of sensitized RBC was about 3 hrs. Since it is believed to be an association between the rate of RBC clearance by anti-D and the ability of the antibody to suppress the immunization, the monoclonal immunoglobulin may prove suitable for immunoprophylaxis of RhD hemolytic disease.

[Monoclonal antibodies against D-antigen of the rhesus system]. / Monoklonal'nye antitela k D-antigenu sistemy rezus.

The authors review current data on the production and characterization of anti-D monoclonal antibodies. The main methods for establishment of anti-D antibody secreting cell lines are considered: EBV-transformation of B lymphocytes from immune donor and hybridization with malignant human or murine cell lines. Serologic and immunological properties of anti-D monoclonal reagents for blood group Rh-typing as well as feasibility of using anti-D monoclonal antibodies as a modality against hemolytic disease of the newborn are discussed.

[Anti-Rho[D] antigen monoclonal antibodies. II. Serological characteristics of monoclonal IgG1 antibodies]. / Monoklonal'nye antitela k Rho[D]-antigenu sistemy rezus. II. Serologicheskaia kharakteristika monoklonal'nykh IgG1-antitel.

Four IgG1 monoclonal antibodies (MoAbs) to the Rh antigen D, produced by stable Epstein-Barr virus transformed B-lymphoblastoid cell lines were assessed in different serological tests. Their suitability in blood group typing was shown in antiglobulin, enzyme or albumin methods. The specificity and activity of MoAbs was tested with a panel of red cells of various Rh-phenotypes. The supernatants of all four lines showed anti-D specificity and ability to react with Du red cells. Mean level of MoAbs concentration was near 10 micrograms/ml. These human anti-D MoAbs proved to be useful D-typing diagnostic reagents.

[Epitope specificity of hemagglutinating monoclonal anti-B antibodies]. / Epitopnaia spetsifichnost' gemaggliutiniruiushchikh monoklonal'nykh anti-B-antitel.

Fine epitope specificity of ten monoclonal antibodies (MA) agglutinating red blood cells B was studied. Three methods were used: 1) inhibition of MA binding to natural antigen by synthetic oligosaccharides (OS) and their polyacrylamide conjugates, 2) direct MA binding to a series of synthetic OS-polyacrylamide conjugates differing in carbohydrate epitope density, 3) direct MA binding to the affinity sorbents. It is shown that all antibodies studied prefer trisaccharide B determinant Gal alpha 1-3(Fuc alpha 1-2) Gal independently of their ability to discriminate serological subgroups of B erythrocytes (B, Bweak, B3). The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells B subgroups is discussed. Of an interest is that MAs which are able to agglutinate any B subgroups also bing the synthetic tetrasaccharide Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc, a B type 3 determinant.