Characterization of a novel anti-human TNF-α murine monoclonal antibody with high binding affinity and neutralizing activity

摘要

In order to develop an anti-human TNF-α mAb, mice were immunized with recombinant human TNF-α. A murine mAb, TSK114, which showed the highest binding activity for human TNF-α was selected and characterized. TSK114 specifically bound to human TNF-α without cross-reactivity with the homologous murine TNF-α and human TNF-β. TSK114 was found to be of IgG1 isotype with κ light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-α by surface plasmon resonance technique revealed a binding affinity (KD) of ~5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, espectively. TSK114 neutralized human TNF-α-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-α-neutralizing antibody with picomolar affinity.