Anti-human DR5 Monoclonal Antibody Research Articles

Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody and the adenovirus encoding soluble TRAIL in prostate cancer

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces a death signal following binding to death receptors (DR4, DR5). We have developed a novel anti-human DR-5 monoclonal antibody (TRA-8) and adenoviral encoding TRAIL (Ad/TRAIL). Herein, we are testing the combined effect of radiotherapy and TRA-8 or Ad TRAIL in prostate cancer cells. Human prostate cancer cell lines LnCap, PC-3 and DU145 were used in this study. Cells were treated either with TRA-8 alone or Ad/TRAIL, radiation alone, or a combination of each at different doses and intervals. Cell survival using the MTS assay and colony forming assay were used to determine radiosensitization. Immunohistochemistry was used to detect bax and bcl-2. Real-time PCR was performed on mRNA of treated prostate cancer cell lines. Finally, a murine model of subcutaneous prostate cancer was used to evaluate the in vivo effect. Cell survival assays detected by MTS assay showed that prostate cell lines treated with a combination of radiation and TRA-8 showed significantly lower survival than cells treated with either radiation or TRA-8 alone. Colony forming assay and cell proliferation assays showed increased killing after combination treatment with TRA-8 or Ad/TRAIL and radiation, than either single agent alone. Mechanistic studies showed that the killing effect was due to induction of apoptosis mostly by increased expression of bax in TRA-8 or Ad/TRAIL treated cells. Additionally, RT-PCR showed an increased copy number of bax in most cells treated with TRA-8 and radiation. It is concluded that radiation and TRA-8 or Ad/TRAIL produced a synergistic effect in refractory prostrate cancer.

Structural basis of LaDR5, a novel agonistic anti-death receptor 5 (DR5) monoclonal antibody, to inhibit DR5/TRAIL complex formation

BackgroundAs a member of the TNF superfamily, TRAIL could induce human tumor cell apoptosis through its cognate death receptors DR4 or DR5, which can induce formation of the death inducing signaling complex (DISC) and activation of the membrane proximal caspases (caspase-8 or caspase-10) and mitochondrial pathway. Some monoclonal antibodies against DR4 or DR5 have been reported to have anti-tumor activity.ResultsIn this study, we reported a novel mouse anti-human DR5 monoclonal antibody, named as LaDR5, which could compete with TRAIL to bind DR5 and induce the apoptosis of Jurkat cells in the absence of second cross-linking in vitro. Using computer-guided molecular modeling method, the 3-D structure of LaDR5 Fv fragment was constructed. According to the crystal structure of DR5, the 3-D complex structure of DR5 and LaDR5 was modeled using molecular docking method. Based on distance geometry method and intermolecular hydrogen bonding analysis, the key functional domain in DR5 was predicted and the DR5 mutants were designed. And then, three mutants of DR5 was expressed in prokaryotic system and purified by affinity chromatograph to determine the epitope of DR5 identified by LaDR5, which was consistent with the theoretical results of computer-aided analysis.ConclusionsOur results demonstrated the specific epitope located in DR5 that plays a crucial role in antibody binding and even antineoplastic bioactivity. Meanwhile, revealed structural features of DR5 may be important to design or screen novel drugs agonist DR5.

Synergistic cytotoxic effect of anti-human DR5 monoclonal antibody hCTB006 and irinotecan on gastric cancer BGC823 and SGC7901 cells

Synergistic cytotoxic effect of anti-human DR5 monoclonal antibody hCTB006 and irinotecan on gastric cancer BGC823 and SGC7901 cells

Treatment of small cell lung cancer with TRA-8 in combination with cisplatin and radiation

BackgroundLimited stage small cell lung cancer (SCLC) represents a minority of SCLC. Despite extensive clinical trials, standard treatment remains cisplatin-based chemotherapy and thoracic irradiation (TI). This study focused on the interaction of cisplatin/radiation with the anti-human DR5 monoclonal antibody TRA-8 in SCLC cells. TRA-8 binds specifically to DR5 and has been shown to activate apoptosis. MethodsFour human SCLC cell lines were utilized for experimentation (SCLC-41, SCLC-58, SCLC-68, and SCLC-74). Immunoblot analysis was used to determine relative protein levels of DR5, DR4 and pro-caspase 8 for each cell line. Using a tetrazolium-based assay (XTT), the IC50 values for cisplatin with or without TRA-8 were determined for the SCLC cell lines. Four SCLC lines were assayed with a combination of TRA-8 (10μg/ml), 2Gy radiation and various concentrations of cisplatin. Apoptosis was evaluated using Annexin V-FITC and cleaved caspase immunoblotting. Using a SCLC-58 subcutaneous xenograft model, treatment began 21d after tumor cell injection. Treatment included weekly cisplatin (4mg/kg) and radiation of 1Gy (24h after cisplatin) and TRA-8 (200μg) was administered i.p. twice weekly for three weeks. ResultsImmunoblot analysis showed similar levels of DR5 for all cell lines with variable levels of DR4. Various concentrations of TRA-8 antibody (⩽10μg/ml) induced no significant cytotoxicity in the SCLC cell lines. The in vitro combination treatment with TRA-8 (10μg/ml), 1.25μg/ml cisplatin and 2Gy radiation showed increased cytotoxicity when compared to combinations without TRA-8. Furthermore, the triple combination demonstrated the greatest amount of apoptosis as measured by Annexin V staining. The in vivo studies showed the combination of 1Gy, cisplatin and TRA-8 extended the tumor doubling time to 44d as compared to any doublet treatment groups that ranged from 12 to 20d. Analysis of survival data showed 100% of the combination group (RT+cisplatin+TRA-8) were alive 65d after treatment began whereas all doublet treatment groups showed 50% or less survival. ConclusionsThese studies showed increased cytotoxicity when TRA-8 was added to radiation/cisplatin in SCLC. This effect was demonstrated in vitro and in vivo. TRA-8 represents a promising new agent in the treatment of SCLC.

Targeting a Novel N-terminal Epitope of Death Receptor 5 Triggers Tumor Cell Death

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

The Construction and Expression of a Novel Chimeric Anti-DR5 Antibody

Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) could selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Unfortunately it has been reported that some forms of TRAIL could cause damage to normal liver cells. In order to avoid this side effect, many groups are now developing agonistic monoclonal antibody (MAb) against DR5. Previously, we developed a MAb (mDRA6) against DR5 with apoptosis inducing ability. The application of mouse origin antibody is limited in clinical use because it can induce human anti-mouse antibody (HAMA) responses that can cause allergic reaction and damage in the human body. Therefore, the variable region genes of this MAb were cloned and ligated into chimeric antibody expression vector (pCMV-VH and pCMV-VL), generating chimeric anti-human DR5 MAb (cmDRA6) expression vectors. The two plasmids were introduced into 293T cells with Lipofectamine 2000. The cell culture supernatant was collected and the expression level of cmDRA6 was detected with standard ELISA method. The chimeric mDRA6 could bind to its target antigen as demonstrated by the results of both ELISA and Western blot. This study of mDRA6 has laid the solid foundation for further application research in the future.

Fas ligand and anti-human DR5 monoclonal antibody induce colon cancer cell line HT29 apoptosis

Objective To study the cytotoxic effects of Fas ligand and anti-human DR5 mono-clonal antibodies (Anti-DR5 mAb) on colon cell line HT29 and the underlying mechanism. Methods Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/Anti-DR5 mAb on tumor cell lines was measured by MTr colorimetry and the induced apoptosis was determined by agarose gel electro-phoresis. The cell cycle and apoptosis was assessed by flow cytometry with PI staining. Caspase-3 and bcl-2 expression was assayed by Westem blot. Results The expression of Fas mRNA in HT29 cells was lower than DR5 mRNA. HT29 cells were sensitive to anti-DR5 mAb but partially sensitive to FasL in a dose de-pendent manner. The killing rate of 50 mg/L FasL and anti-DR5 mAb to HT29 cells was (25.49± 0, 90)% and (48.90 ± 3.15 )%, respectively. The cell cycle analysis suggested that FasL/ Anti-DR5 mAb could inhibit HT29 cells in G0/G1 phase, and then further inhibit cell growth. The apoptosis rate of 25 mg/L FasL and anti-DR5 mAb to HT29 cells was ( 13.8±1.5 ) % and (22.6±1.1 ) % ,respectively. The expression of Caspase-3 was increased, but the expression of bcl-2 was decreased obviously in HT29 cells after treated with FasL/Anti-DR5 mAb. Conclusion FasL/ Anti-DR5 mAb could induce apoptosis of HT29 cells. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, and the release of Caspase-3 and bcl-2. Key words: Colon carcinoma; Death receptor; Apoptosis

A Novel Anti-human DR5 Monoclonal Antibody with Tumoricidal Activity Induces Caspase-dependent and Caspase-independent Cell Death

Like anti-Fas monoclonal antibodies, some monoclonal antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors have tumoricidal activity too. In this article we report a novel mouse anti-human DR5 monoclonal antibody, AD5-10, that induces apoptosis of various tumor cell lines in the absence of second cross-linking in vitro and showed strong tumoricidal activity in vivo. AD5-10 does not compete with TRAIL for binding to DR5 and synergizes with TRAIL to induce apoptosis of tumor cells. AD5-10 induces both caspase-dependent and caspase-independent cell death in Jurkat cells, whereas TRAIL induces only caspase-dependent cell death. We show for the first time that DR5 can mediate caspase-independent cell death, and DR5 can mediate distinct cell signals when interacting with different extracellular proteins. Studies on AD5-10 help us to understand more on the functions of DR5 and may provide new ideas for cancer immunotherapy.

Detection of disseminated breast cancer growth and treatment response using non-invasive bioluminescence imaging

742 Background: TRA-8, a mouse anti-human DR5 monoclonal antibody induces apoptosis in TRAIL-sensitive tumor cells. However no non-invasive method is available to monitor the treatment effects on disseminated cancer. We report a non-invasive and sensitive BI technology to determine the efficacy of combined treatment of TRA-8 and Adriamycin on disseminated breast cancer in animal model. Methods: Human breast cancer cell line 2LMP, a subclone of MDA-MB-231, was transfected with adeno associated virus encoding luciferase (AAV-luc). A stable luciferase-positive cell line was established by screening. Two groups (5/group) of athymic female nude mice were used. Four sites (liver, spleen, chest cavity and peritoneum) of each mouse were injected with luciferase-positive 2LMP cells (0.25x106/site). After 7 days, BI revealed disseminated tumor sites; one group of mice was injected i.v. with 150 μg TRA-8 and 6 mg/kg Adriamycin, and the 2nd group of mice did not receive any treatment. All treatments were given 2X/wk ...

Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody in prostate cancer

Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody in prostate cancer

Bisindolylmaleimide VIII Enhances DR5-mediated Apoptosis through the MKK4/JNK/p38 Kinase and the Mitochondrial Pathways

Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy.

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.