RESUMO
BACKGROUND: The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and non-plasma-derived source of antibodies for Rhesus prophylaxis is needed. STUDY DESIGN AND METHODS: Recombinant human immunoglobulin G (IgG)1, IgG2, IgG3, IgG4, IgA1, and IgA2 anti-D with the same variable region were expressed in Chinese hamster ovary cells. The effector functions of these antibodies were assessed by an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and a chemiluminescence (CL) method for detection of respiratory burst. RESULTS: In the ADCC assay, IgG1, IgG3, and IgA1 did the best and were as active as a currently used prophylactic polyclonal anti-D. IgG4 and IgA2 were moderately active, whereas IgG2 was not active. In the CL assay, IgG1 and IgG3 were active but much less so than a currently used prophylactic polyclonal anti-D. For some effector cell preparations, IgG4 was active in the CL assay, whereas IgG2, IgA1, and IgA2 were not. A mixture of IgG1 and IgG3 showed a synergistic effect in the CL assay and did as well as the prophylactic polyclonal anti-D in ADCC and CL. Mixtures of IgA1 and either IgG1 or IgG3 showed no synergistic effect. CONCLUSION: A mixture of recombinant human IgG1 and IgG3 anti-D could be of value in future Rhesus prophylaxis.
Assuntos
Imunoglobulina A/genética , Imunoglobulina G/genética , Proteínas Recombinantes/genética , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Expressão Gênica/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Técnicas In Vitro , Proteínas Recombinantes/imunologia , Explosão Respiratória , TransfecçãoRESUMO
For over 35 years hemolytic disease of the fetus and newborn (HDFN) due to RhD has been effectively prevented by anti-RhD antibodies obtained from alloimmunized women or deliberately immunized men. However, due to the reduced number of immunized women and for ethical reasons it is foreseen that other sources of anti-RhD will be needed. One such source is recombinant human antibodies. Here we describe the construction of plasmids encoding two subclasses (IgG1 and IgG3) of an anti-RhD antibody, their transient expression in COS cells, and subsequent functional characterization of the antibodies with regard to specificity and ability to mediate a respiratory burst. The recombinant anti-RhD antibodies were specific for the RhD antigen and were able to mediate a respiratory burst. Thus these antibodies might be of use as future rhesus prophylaxis.
Assuntos
Células COS/metabolismo , Engenharia de Proteínas/métodos , Transfecção , Animais , Especificidade de Anticorpos , Chlorocebus aethiops , Eritroblastose Fetal/prevenção & controle , Humanos , Imunoglobulina G/análise , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Medições Luminescentes , Peptídeos/imunologia , Plasmídeos/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Explosão Respiratória , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Imunoglobulina rho(D)/análise , Imunoglobulina rho(D)/biossíntese , Imunoglobulina rho(D)/genética , Imunoglobulina rho(D)/imunologiaRESUMO
More than 100 million individuals exhibit IgE-mediated allergic reactions against Phl p 2, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phl p 2-specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen-allergic patient. Recombinant Phl p 2-specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N-terminal amino acids of Phl p 2 and cross-reacted with group 2 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG(1) to obtain a complete, recombinant Phl p 2-specific human IgG(1). This antibody blocked the binding of grass pollen-allergic patients IgE (n=26; mean inhibition: 58%) to Phl p 2 and caused a 100-fold reduction of Phl p 2-induced basophil histamine release. The recombinant human Phl p 2-specific IgG(1) may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.
Assuntos
Alérgenos/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Reações Cruzadas , Liberação de Histamina , Humanos , Imunoglobulina E/genética , Imunoglobulina G/genética , Engenharia de ProteínasRESUMO
To establish stable cell lines that produce recombinant multisubunit proteins, it is usually necessary to cotransfect cells with several independent gene constructs. Here, we show that a stepwise fusion of individually transfected cells, results in a fused cell-line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a defined expression ratio between each protein subunit. The cell-fusion technology described allows a predefined expression level of each subunit. Using SIgA as a model protein we demonstrate that the majority of the fused cells inherit the molar expression ratio of the parental transfected cells. These results indicate that simplified screening of clones expressing the expected subunit ratios may be possible using the cell-fusion technology. This technology may therefore be an alternative to generic transfection methods for the establishment of cells that produce multiprotein complexes such as antibodies, receptors, ion channels and other multisubunit proteins.
Assuntos
Fusão Celular/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Animais , Células CHO , Cricetinae , Imunoglobulina A/genética , Imunoglobulina A/metabolismo , Subunidades Proteicas , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Antibodies can neutralize the infectious properties of human cytomegalovirus (CMV). In vivo, the major neutralization determinants are located on glycoprotein B (gB). Recombinant human antibodies, that carry different constant regions (IgG1, IgG3 and the synthetic variant IgG3mA) against two of these epitopes were investigated for their ability to recruit the complement cascade for destruction of the virus. It was shown that all variants of an antibody against the antigenic domain (AD)-2 epitope displayed a similar neutralization activity despite the fact that improved C1q binding was observed for IgG3 and IgG3mA over the IgG1 variant. In contrast, an antibody against the AD-1 epitope carrying the normal IgG3 constant region, was less efficient than its IgG1 counterpart in neutralizing the virus in the absence of complement. However, it restored its activity in the presence of complement to the level of the naturally occurring IgG1 version. The same antibody was substantially more potent in neutralizing the virus in the presence of complement if it carried the IgG3mA constant region. This demonstrates the importance of the constant domain for the biological activity of AD-1 specific antibodies, a factor that should be taken into account when using antibody-based therapeutics or when inducing antibodies by vaccination.