RESUMEN
A monoclonal human IgG1, Campath-1H, was digested with glycosidases to assess the effect of carbohydrate on the functional activities of an IgG1. Removal of the complete carbohydrate moiety abolished complement lysis activity and antibody-dependent cell-mediated cytotoxicity, but left antigen binding activity and protein A binding activity intact. Removal of terminal sialic acid residues through glycopeptidase F digestion was not found to affect any of the tested IgG activities. Removal of the majority of the galactose residues from desialylated Campath-1H was found to reduce but not abolish complement lysis activity. Other activities were not affected by degalactosylation. This indicates a rare separation of complement lysis activity and antibody-dependent cell-mediated cytotoxicity of IgG in the way they behave under controlled conditions. This paper underlines the overall importance of carbohydrate in IgG function and stresses the relative contributions of some of the carbohydrate residues.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/fisiología , Anticuerpos Antineoplásicos/metabolismo , Anticuerpos Antineoplásicos/fisiología , Galactosa/metabolismo , Ácidos Siálicos/metabolismo , Alemtuzumab , Amidohidrolasas/farmacología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Anticuerpos Antineoplásicos/química , Citotoxicidad Celular Dependiente de Anticuerpos , Células CHO , Secuencia de Carbohidratos , Proteínas del Sistema Complemento/fisiología , Cricetinae , Pruebas Inmunológicas de Citotoxicidad , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Inmunoglobulina G/fisiología , Datos de Secuencia Molecular , Mieloma Múltiple/inmunología , Ácido N-Acetilneuramínico , Neuraminidasa/farmacología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A new and faster assay for antibody dependent cellular cytotoxicity based on release of europium from target cells is described. This has a number of important advantages over the traditional assays based on release of chromium-51 (51Cr). The new method involves labelling of Wein 133 target cells (B cell non-Hodgkin's lymphoma cells) which express the antigen, CDw52, with the chelate europium diethylenetriaminopentaacetic acid (EuDTPA) according to the method of Blomberg et al. (1986). Labelled cells are sensitised (coated) with the anti-lymphocytic monoclonal antibody, Campath-1H. Human peripheral blood mononuclear cells are added to mediate lysis of EuDTPA labelled Wein 133 cells by ADCC. Release of EuDTPA from lysed cells is determined by mixing supernatants with enhancement solution containing 2-naphthoyl trifluoroacetone, 2-NTA, to form a highly fluorescent chelate which is measured using time resolved fluorometry. Results obtained with the new EuDPTA release assays were comparable to traditional assays based on the release of the radioisotope 51Cr. It is anticipated that this assay will have a widespread application among laboratories performing ADCC assays. The method is non-hazardous and has been used routinely for over 2 years to monitor production and purification of Campath-1H.