Fusion protein of CDR mimetic peptide with Fc inhibit TNF-α induced cytotoxicity

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-α (TNF-α) neutralizing monoclonal antibody (named as Z12) with TNF-α, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-α. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-α induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.