The tetravalent anti-DR5 antibody without cross-linking direct induces apoptosis of cancer cells.

ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand can induce apoptosis in many tumor cell lines. This apoptotic effect is mediated by interaction of TRAIL and its receptors, which include Death Receptor 4 (DR4) and Death Receptor 5 (DR5). Some antibodies to DR4 or DR5 do not have anti-tumor ability without cross-linking but exhibit anti-tumor ability in the presence of a cross-linking reagent. Here, we suggest that the tetravalent anti-DR5 antibody can induce apoptosis of cancer cells independent of cross-linking reagent. The single-chain variable fragment of the anti-DR5 antibody, HSA (human serum albumin) - p53 gene, comprising residues 490-513 of HSA and the tetramerization domain of human p53 were assembled into the tetravalent antibody by an overlapping PCR. Results of size exclusion HPLC indicated that the purified protein exhibited a major peak (tetramer) and a minor peak (dimer). MTT assay demonstrated the tetravalent antibody without cross-linking could inhibit survival of Jurkat and EC9706 cells in a dose-dependent manner while the monovalent antibody could not inhibit survival of Jurkat and EC9706 cells. IC50 of Jurkat cell was 3.2 mg/L and IC50 of EC9706 cell was 3.9 mg/L. Furthermore, the Annexin V/PI assay and the Hoechst 33258 staining showed that the tetravalent antibody could efficiently induce apoptosis of Jurkat and EC9706 cells. Therefore, the tetravalent anti-DR5 antibody can act as a direct agonistic antibody, and initiate efficient apoptotic independent of cross-linking reagent. Thus, the tetravalent anti-DR5 antibody will be a new kind of candidate for potential cancer therapeutics.