No abstract

The extracellular domain of tumour necrosis factor (TNF) receptor II fused with the human IgG1 Fc region (TNFRII-Fc), as well as antibodies against TNF, has been used to treat rheumatoid arthritis. However, TNFRII-Fc is less effective than these antibodies in terms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against cells bearing TNF on the cell surface. We hypothesized that these activities could be increased by fusing TNFRII with tandemly repeated Fc (TNFRII-Fc-Fc). The affinities of TNFRII-Fc-Fc for soluble TNF-α and transmembrane TNF-α and the TNF-α cytotoxicity-inhibitory activity were as potent as those of TNFRII-Fc. TNFRII-Fc-Fc showed much higher binding avidity for Fcγ receptors than TNFRII-Fc and was more potent in terms of both ADCC and CDC against cells expressing transmembrane TNF-α. TNFRII-Fc-Fc of 80 kDa, as well as TNFRII-Fc-Fc of 200 kDa, was detected. TNFRII-Fc-Fc (80 kDa) was as potent as TNFRII-Fc in terms of both ADCC and CDC. These results suggest that Fc multimerization of receptor-Fc fusion proteins can augment effector functions such as ADCC and CDC, and thereby have the potential to provide a superior therapeutic effect. This may be the case not only for TNFRII-Fc but also for other receptor-Fc fusion proteins.

The Fc domain of human IgG1 binds to Fcc receptors (FccRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FccRIIA and FccRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH 2 CONH 2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FccRI and FccRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cellmediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FccR expression levels on the THP-1 cells was FccRII > FccRI > FccRIII and ADCP was inhibited by blocking antibodies against FccRI and FccRII. These results imply that the effect of the interchain disulfide bond cleavage on FccRs binding and ADCP is dependent on modifications of the cysteine residues and the FccR isotypes.