Renewal of EBV-hybridoma method: Efficient generation of recombinant fully human neutralizing IgG antibodies specific for tetanus toxin by use of tetroma cells

Abstract

We have generated tetroma cell lines by the fusion of newly developed parental 6JC5.2 cells with a human B cell line, which was transformed with Epstein-Barr virus (EBV) and enriched with antibody-forming cells that produce neutralizing antibodies to tetanus toxin (TT) by a limiting dilution method using IL-6. The resultant two tetroma cell lines stably produced different monoclonal antibodies (mAbs), TT1 (IgG1·λ) and TT2 (IgG4·κ) reactive with TT after three-times consecutive cell cloning. Although weak to almost nonexistent neutralizing activities against TT were detected in TT1 and TT2 mAbs, respectively, mixing of them resulted in a dramatic increase in the neutralizing activity and complete protection from the toxin was observed in vivo. Moreover, functional immunoglobulin (Ig) genes were cloned from at least 10 cells in the first cloning step of tetromas after the cell fusion. None of the endogenous Ig genes, derived from the parental cell that hinders functional Ig gene cloning, was amplified. In addition, the EBV genome derived from the B cells was eliminated from the antibody producing tetroma lines. This classical but revised EBV-hybridoma method using fusion partner 6JC5.2 may become one alternative method for production of fully human antibodies useful for prevention and treatment of infectious diseases and cancer.