[Expression and antigen binding activity of anti-human CD86 diabody]

To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary (CHO) cells, then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect.

The antibody heavy and light chain viable region gene (V(H); and V(L);) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody. Anti-human CD86 diabody gene V(H);-(GGGGS)-V(L); was constructed by SOE-PCR. Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody. The recombinant vector was transfected into CHO cells with Lipofectamine(TM); 2000, and the cell clones secreting CD86 diabody were screened by G418. We used IMAC to purify CD86 diabody and quantified its concentration by BCA method. The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry. After Raji cells were treated with CD86 diabody for 72 h, its proliferation inhibiting effect was investigated by MTT assay.

We have obtained one CHO cell line that stably secreted CD86 diabody. The concentration of CD86 diabody after purification was 5.24 mg/L. The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%, respectively. After CD86 diabody treatment for 72 h, the inhibition rate of Raji cells was 37%.

The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically, and inhibit the proliferation of these tumor cells effectively.