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. 2010 Sep-Oct;2(5):565-70.
doi: 10.4161/mabs.2.5.12570. Epub 2010 Sep 1.

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

Yang Feng  1 , Xiaodong Xiao, Zhongyu Zhu, Dimiter S Dimitrov
Affiliations

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

Yang Feng et al. MAbs. 2010 Sep-Oct.

Abstract

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.

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Figures

Figure 1
Figure 1
Selection of DR4-specific antibodies from human phage-displayed Fab library. (A) Diagram of the recombinant DR4 protein used for selection. (B) In vitro binding of the two selected antibodies to DR4, m921 IgG and m922 IgG. The two IgGs were 1:5 serially diluted from 1,000-0.0128 nM.
Figure 2
Figure 2
Competition of m921 and m922 with rTRAIL and other antibodies for DR4 binding in vitro. (A) DR4 was first coated on ELISA plate, m921 or m922 IgG, at 10 nM was pre-incubated with various concentrations of soluble recombinant TRAIL (rTRAIL), before they were added to ELISA wells. Binding of IgG was detected and plotted against different concentrations of competing rTRAIL. (B) m921 or m922 IgG was incubated with various concentrations of competing mouse/rabbit IgG, before they were added to ELISA wells with DR4 protein coated. Binding of m921 or m922 was detected. (C) Each competing antibody was tested separately at concentrations from 0.8 nM to 100 nM to confirm their binding to DR4.
Figure 3
Figure 3
Flow cytometry of DR4 binding to m921 and m922 on ST486 cells. ST486 cells were incubated with 24 nM primary antibodies for 1 h on ice, followed by 2 ul of corresponding FITC-conjugated secondary antibodies for 30 min. Black line, a control human monoclonal Ab. Green line, DR4 mouse monoclonal antibody from Santa Cruz Biotechnology. Red line, DR4 IgG m921. Blue line, DR4 m922 IgG.
Figure 4
Figure 4
Growth of ST486 in the presence of DR4 m921 and m922. ST486 cells were seeded in clear 96-well culture plates at 3,000 cells/well. Treatment media with antibodies were added to wells. m921 or m922 IgG concentrations are at 10, 50, 250 or 500 nM in the final culture samples. Cells were treated with 5 nM of soluble recombinant TRAIL (rTRAIL) as positive control. An isotype control IgG at 500 nM was included as negative control (C). Cell viability was monitored 48 hours after the treatment. Six wells were set up for each treatment. The graph shown here is a representative result. Readings from each treatment were compared with those from the control group in a t-Test analysis, *in graph stands for p < 0.05 and ** stands for p < 0.01.
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