Abstract

According to the American Brain Tumor Association, primary brain tumors occur at a rate of 12.8 per 100,000 people. However, tumors growing in the brain are difficult to treat. Now, researchers have found that antibodies utilize complementarity-determining regions (CDRs) of their variable domains to bind antigens with high affinity and specificity. Therefore, we established an antibody mimetic fused with diphtheria toxin for targeting glioma cell line of U87 MG. V_HCDR1 and V_LCDR3, together with 5 amino acid residues on both side of the CDRs, through a cognate framework region (V_HFR2) yielded a mimetic of BT32/A6 (United States Patent number: 5639863). We fused the mimetic with the first 388 amino acid residues of diphtheria toxin through a linker of GGGS. Escherichia coli strain BL21 (ED3) was used to express the soluble immunotoxin DTLMG. The killing activity of U-87 MG cellsby this fusion protein was examined in vitro. The immunotoxin DTLMG alone did not kill Raji up to the maximal concentration tested (10^-6M) in vitro. By contrast, concentrations ≥10^-9M, of the fused DTLMG killed more than 95% of U-87 MG cells. It is suggested that the mimetic maintained the synergic interactions and high-affinity associated with the parent antibody. This construct holds promise for targeting aimed cancer epitopes and raised hopes for their more effective and much wider use in the future.

Key words: Glioma cell, fusion protein, U87 MG.

Abbreviation

Abbreviations: CDRs, Complementarity-determining regions; mAbs, monoclonal antibodies; FRs, framework regions; DNA, deoxyribonucleic acid; PI, propidium Iodide; VH, heavy chain; VL, light chain.