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

Abstract

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, respectively. 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.

A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes

“…Three days after the last injection, spleen cells from the immunized mice were fused with murine myeloma Sp2/O cells (American Type Culture Collection) in the presence of 50% (w/v) polyethylene glycol 1500 (Roche; ref. 25). From the screening of hybridoma supernatants against DR4-ECD using ELISA, 10 positive hybridomas were isolated ( Supplementary Fig.…”

“…AY4 was purified from the cell supernatant by Protein A-Sepharose 4 Fast Flow (Pharmacia; ref. 25). Cloning of AY4 gene from the hybridoma by reverse transcription-PCR and then sequence analysis revealed that the subclass of AY4 mAb was IgG1 for heavy chain and κ for light chain.…”

Interrelationships between Infliximab and Recombinant Tumor Necrosis Factor-αin Plasma Using Minimal Physiologically Based Pharmacokinetic Models

“…The binding association rate constant (k on ) of infliximab was fixed to 10 5 M 21 × S 21 (Kim et al, 2007;Song et al, 2008).…”

Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo

“…Of the 4 TNFα binding KD variants, biological activity assays using a human TNFα-responsive WEHI 164 cell bioassay (20,23) revealed that only KDT26 neutralized TNFα-mediated cytotoxicity in a concentration-dependent manner, showing IC 50 (50% inhibitory concentration) of ∼0.8 μM (Fig. 4A).…”

“…In vitro and in vivo neutralizing activities of PgnKD2, the KD variants, KDT26-Fc, and/or infliximab were measured against human TNFα-induced cytotoxicity using mouse WEHI 164 cells (ATCC) and TNFα-induced lethality in C57BL/6 mice (Orientbio), as described previously (20,23,24).…”