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Characterization of stable Epstein-Barr (EB) virus transformed cell lines and mouse-human hybridomas producing a large quantity of anti-tetanus toxoid (TT) monoclonal antibody.

Antigen-specific human monoclonal antibodies (hMoAb) were investigated with particular emphasis on the stability and scale-up of production. Peripheral blood lymphocytes from a healthy donor previously vaccinated with tetanus toxoid (TT) were resensitized in vitro with the antigen in the presence of pokeweed mitogen (PWM). Three days after the stimulation, lymphocytes were infected with Epstein-Barr (EB) virus. Among EB virus transformants, several lymphoblastoid cell clones producing anti-TT hMoAb were established. One of these clones, 3G6, produced about 10 micrograms/ml of IgM (K) in the culture supernatant. Furthermore, 3G6 cells were fused with murine myeloma cells (X63-Ag8 X 653) and three heterogeneous hybridoma clones (HE719, HE810 and HE811) were obtained after careful hybrid selection in hypoxanthine-aminopterin-thymidine-ouabain containing medium and cloning by limiting dilution technique. These hybrid clones contained human HLA and mouse H-2d antigens together with mouse-human mixed karyotype. When these mouse-human hybridomas were injected i.p. into pristane-treated nude mice of BALB/c origin, ascites were found and a large amount of anti-TT hMoAb up to 0.5 mg/ml were produced there. These hybridomas have retained their anti-TT antibody-producing capabilities over 10 months. The approach described here has a potential application for the production of other antigen-specific hMoAb.