Anti-Tumor Study Of Multivalent Anti-DR5 Antibody

IntroductionA plethora of studies indicates that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in transformed and cancer cells originating from multiple tissues and organs. This apoptotic effect is mediated by interaction of TRAIL and its receptors, which include TRAIL-R1(DR4) and TRAIL-R2(DR5). Disappointedly, translation of this apoptosis-inducing ability of TRAIL on malignant cells into clinical application has been frustrated by its off-targeting effects on human normal cells, in particular, normal liver cells.Interestingly, some clones of monoclonal antibodies against TRAIL-death receptor can resemble the apoptotic effects of TRAIL without adverse effects on human normal liver cells. Based on our previously established monoclonal antibody against DR5, i.e.YM366EC, we cross-linked the variable region, VL and VH of DR5 antibody and generated one multivalent anti-DR5 antibody, which strikingly suppressed the proliferation of a variety of lines of tumor cells and induce apoptosis on TRAIL-sensitive cells. However, monoclonal antibody has inherent restraints which precludes its translation into bedside application, e.g. human anti-mouse antibody reaction (HAMA) leading to immunologic rejection, big molecular weight, etc. With the advent of DNA recombination technology, it is possible to design and produce gene engineering antibody, which is able to retain the activity of monoclonal antibody, such as homogeneity, specificity, and importantly, overcome the limits of monoclonal antibody of mouse source. Multivalent antibody is a paradigm of gene engineering antibody and is developing rapidly in recent years.ObjectiveTo design and establish a multivalent YM366EC antibody with apoptotic-inducing activity on tumor cells mediated by cross-lining of DR5.MethodsUsing degenerate PCR and 5′-RACE, the variable regions of VL and VH of DR5 antibody were amplified from hybridoma of YM366EC, respectively, and subsequently linked in tandem by using hinge area/C4 binding protein gene of p53 by overlap extension PCR to obtain fused genes for tetramer(3S) and octamer antibody(3B), which was named 3S and 3B. The authenticity of the fused gene was confirmed by sequencing and then was subcloned into eukaryotic expression vector pSecTag2A by HindⅢand XhoⅠrestriction sites, and therefore p-3D, p-3S and p-3B were produced. CHO was infected by individual plasmidsof p-3D, p-3S and p-3B to express fused protein. After purification of expressed proteins by Ni2+-affinity chromatography, SDS-PAGE and western blot was used to verify the experimental molecular weight and reaction with DR5. The anti-tumor and proliferation-inhibiting activity of multivalent antibodies was analysed by flow cytometric analysis and MTT, respectively.ResultsFragments of variable regions of heavy and light chain of anti-DR5 antibody were cloned and then inserted into the eukaryotic expression vector to express the individual unimer, tetramer and octamer protein, respectively. SDS-PAGE and western blot showed that p-3D, p-3S and p-3B expressed protein with molecular weight 35 KD, 40 KD and 40 KD consistent with the theoretic values. Tetramer and octamer antibodies could significantly inhibit the proliferation of tumor cells by MTT assay. Furthermore, flow cytometric analysis showed that tetramer and octamer antibodies displayed potent antitumor activity.ConclusionVariable regions of anti-DR5 antibodies was cloned and subsequently ligated to the eukaryotic expression vector to express unimer, tetramer and octamer antibodies. Function studies showed that multivalent antibodies of tetramer and octamer antibodies have potent antitumor activity, which paved avenue for in vivo studies using our multivalent anti-DR5 antibodies.