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

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta TSK114 was found to be of IgG1 isotype with kappa 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-alpha 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-alpha-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-alpha-neutralizing antibody with picomolar affinity.

Physical and Biological Activity of Domestic Product of Modified Bovine Lung Surfactant

PURPOSE: Neonatal respiratory distress syndrome is caused by the deficiency of lung surfactant in premature babies. For the treatment of RDS at present surfactants such as Surfacten (Tokyo-Tanabe Co., Japan) and Exosurf (Wellcome Co., USA) are used. As awarded the grant from the Ministry of Science and Technology for a model research project of Medium-Technology program, we have modified (supplemented) the bovine lung extracts to get YY-38, for which we have performed physical and biological activities. METHODS: For physical properties, we performed stable microbubble test (SMR) and measured surface tension lowering activity using a pulsating bubble surfactometer. Minimum and maximum surface tensions measured at 1 and 5 minutes gave surface tension-surface area diagrams, from which compressibility at surface tension 10mN/m was also calculated. As to the biological activity, we used premature rabbit fetuses as a model for the study of pressure-lung volume relationship. The lung pathology was examined on the lung tissues subsequently obtained, and aerated area ratios were calculated based on the area measured by an image analyzer. RESULTS: The minimum surface tensions of YY-38 at 1 and 5 minutes for all different concentrations were low at 10mN/m, while the maximum surface tensions ranged from 33.01mN/m to 41.07mN/m. The surface tension-surface area curve showed a definite hysteresis at 1 and 5 minutes for all concentrations, and the surface tension fell below 10mN/m with 20% surface area compression. The compressibilities at surface tension 10mN/m at 5minutes for all concentrations were all below 0.02. In animal experiments, the mean lung volume of premature rabbit fetuses was inflated to 80.9ml/kg at maximum 30cmH2O, while the lung volume was maintained at 38.3mg/kg when the lung was deflated to 5cmH2O. The overall aerated area ratio was 45.4%. CONCLUSIONS: YY-38 formed sufficient amount of stable microbubbles and had a surface tension low enough to maintain alveolar stability and to exhibit a good hysteresis curve. In animal experiments it helped the expansion of premature lungs during inspiratory phase and was effective in the prevention of collapse during expiratory phase.