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

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.     

Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signaling through the DR4 receptor by competing for ligand binding

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a "dominant negative" effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Myc Tagging along the TRAIL to Death Receptor 5

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL; also known as Apo2L) is an apoptotic cytokine that is being developed as a novel anticancer agent. TRAIL mediates its effect via death receptors 4 (DR4) and DR5 and appears to selectively induce apoptosis in cancer cells. The molecular basis of why normal cells seem to better tolerate this novel cytokine remains unknown. Recently, it has been reported that Myc oncoprotein by upregulating DR5 appears to augment cellular susceptibility to TRAIL and to DR5 agonistic antibodies. Several previous studies have already established that various clinically relevant agents by upregulating DR5 sensitize cells to TRAIL. However, the finding that DR5 is upregulated by an oncoprotein that is overexpressed in several tumor types is noteworthy and may spark future investigations aiming to explore the Myc and DR5 expression status of primary tumors and their ultimate vulnerability to DR5-targeted therapeutics.     

Combination of TRAIL with Bortezomib Shifted Apoptotic Signaling from DR4 to DR5 Death Receptor by Selective Internalization and Degradation of DR4

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.     

Regulation in the targeting of TRAIL receptor 1 to cell surface via GODZ for TRAIL sensitivity in tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promote the selective clearing of various malignancies by inducing apoptosis, holding the promise as a potent therapeutic agent for anticancer. Though DR4 and DR5 have high sequence similarity, differential regulation of both receptors in human tumor cells remains largely unexplored. Here, we repot that golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein (GODZ) regulates TRAIL/DR4-mediated apoptosis. Using the SOS protein recruitment-yeast two-hybrid screening, we isolated GODZ that interacted with the death domain of DR4. GODZ binds to DR4, but not to DR5, through the DHHC and the C-terminal transmembrane domain. Expression level of GODZ affects apoptosis of tumor cells triggered by TRAIL, but not that induced by TNF-α/cycloheximide (CHX) or DNA-damaging drugs. In parallel, GODZ functions to localize DR4 to the plasma membrane (PM) via DHHC motif. Also, introduction of mutation into the cysteine-rich motif of DR4 results in its mistargeting and attenuates TRAIL- or GODZ-mediated apoptosis. Interestingly, GODZ expression is highly downregulated in Hep-3B tumor cells, which show resistance to TRAIL. However, reconstitution of GODZ expression enhances the targeting of DR4 to cell surface and sensitizes Hep-3B cells to TRAIL. Taken together, these data establish that GODZ is a novel DR4-selective regulator responsible for targeting of DR4 to the PM, and thereby for TRAIL-induced apoptosis.     

Localization and variation of TRAIL and its receptors in human placenta during gestation

The localization of TRAIL and its receptors in human placenta was studied under light microscopy using immunohistochemistry method. The variation of TRAIL and its receptors with development was also detected by in situ semi-quantification. The syncytiotrophoblast, cytotrophoblast, stromal cells and the capillary endothelium cells in human placenta all appeared to be TRAIL immunoreactive and the immunoreactive material was distributed on membrane and in cytoplasm with negative nuclei. During whole gestation there was no obvious variation of the staining of TRAIL. Although DR4, DR5, DcR1 and DcR2 can also be detected in the placenta throughout pregnancy, DR4 and DR5 staining increased with development whereas DcR1 and DcR2 staining decreased. Interestingly, at the beginning of the gestation DR4 and DR5 staining distributed on the cytotrophoblast mainly, whereas DcR1 and DcR2 mainly located in the syncytiotrophoblast cells. Collectively, these results suggest that human placenta may not only produce TRAIL but also be a TRAIL target organ, and that TRAIL/TRAILR system could take part in the self-homeostasis of placenta during whole gestation.     

The effect of cisplatin on cytotoxicity of anticancer cytokine TRAIL and its receptor-selective mutant variant DR5-B<Superscript>1</Superscript>

Cytokine TRAIL selectively induces apoptosis in vitro and in vivo in tumor cells without affecting normal cells, but its therapeutic application is limited, since many primary tumors are insensitive to TRAIL. To improve the efficiency of TRAIL, we have previously developed TRAIL mutant variant DR5-B, which binds the apoptosis-inducing death receptor DR5 as efficiently as wild type TRAIL, but shows almost no affinity to other receptors. In this study, we investigated the effect of the chemotherapeutic agent cisplatin on the cytotoxicity of TRAIL variants in 12 tumor cell lines of various origin. Cisplatin effectively enhances the cytotoxic activity of TRAIL preparations. The synergistic effect is most pronounced in the prostate cancer cell lines, where the combined effect exceeds the sum of the separate effects by more than 2 times. The cytotoxicity of DR5-B variant is significantly higher compared to wild-type TRAIL in combination with cisplatin in 9 of 12 tumor cell lines.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

Targeting the extrinsic apoptosis signaling pathway for cancer therapy

The extrinsic apoptosis pathway is triggered by the binding of death ligands of the tumor necrosis factor (TNF) family to their appropriate death receptors (DRs) on the cell surface. One TNF family member, TNF-related apoptosis-inducing ligand (TRAIL or Apo2L), seems to preferentially cause apoptosis of transformed cells and can be systemically administered in the absence of severe toxicity. Therefore, there has been enthusiasm for the use of TRAIL or agonist antibodies to the TRAIL DR4 and DR5 in cancer therapy. Nonetheless, many cancer cells are very resistant to TRAIL apoptosis in vitro. Therefore, there is much interest in identifying compounds that can be combined with TRAIL to amplify its apoptotic effects. In this review, I will provide a brief overview of apoptosis signaling by TRAIL and discuss apoptosis-sensitizing agents, focusing mainly on the proteasome inhibitor bortezomib (VELCADE) and some novel sensitizers that we have recently identified. Alternative ways to administer TRAIL or DR agonist antibodies as therapeutic agents will also be described. Finally, I will discuss some of the gaps in our understanding of TRAIL apoptosis signaling and suggest some research directions that may provide additional information for optimizing the targeting of the extrinsic apoptosis pathway for future cancer therapy.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

TNF-related apoptosis-inducing ligand as a therapeutic agent in autoimmunity and cancer

Recombinant, soluble TNF-related apoptosis-inducing ligand (TRAIL) is currently being developed as a promising natural immune molecule for trial in cancer patients because it selectively induces apoptosis in transformed or stressed cells but not in most normal cells. In cancer patients, phase 1 and 2 clinical trials using agonistic mAbs that engage the human TRAIL receptors DR4 and DR5 have also provided encouraging results. It is now evident that TRAIL suppresses autoimmune disease in various experimental animal models, suggesting that the therapeutic value of recombinant TRAIL and agonistic DR4 and DR5 mAbs might also extend to the suppression of autoimmune disease. This review provides an insight into our current understanding of the role(s) of TRAIL in disease, with a specific focus on cancer and autoimmunity. We also emphasize biological agents and drugs that sensitize tumour cells to TRAIL-mediated apoptosis and discuss the potential molecular basis for their sensitization.     

Cancer: novel therapeutic strategies that exploit the TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway

Cancer is a widespread disease, with half of all men and one-third of all women in the United States developing cancer during their lifetime. The efficacy of many cancer treatments including radiotherapy, chemotherapy and immunotherapy is due to their ability to induce tumor cell apoptosis. Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently being developed as a cancer therapeutic since it selectively induces apoptosis in a variety of transformed cells, but not in most normal cells. Agonistic monoclonal antibodies (mAbs) specific for human death-inducing TRAIL receptors (DR4 or DR5) are also being actively pursued. Importantly, in experimental mice, synergistic anti-tumor effects have been observed with a combination treatment of agonistic mAb against DR5 together with either IL-21 or agonistic mAbs against CD40 and CD137. Together, these findings suggest that antibody-based therapies that cause tumor cell apoptosis and promote T cell memory or function may be effective in fighting cancer.     

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes that TRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL and its receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignant tissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1-4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levels of TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not as widespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAIL cytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role of TRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable to TRAIL therapy.     

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19⁺ lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34⁺ TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.     

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N NMR resonance assignments and secondary structure determination of the extra-cellular domain from the human proapoptotic TRAIL-R2 death receptor 5 (DR5-ECD)

Death receptors (DR) selectively drive cancer cells to apoptosis upon binding to the Tumor necrosis factor-a-Related Apoptosis-Inducing Ligand (TRAIL). Complex formation induces the oligomerization of the death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and transduces the apoptogenic signal to their respective death domains, leading to Death Inducing Signaling Complex (DISC) formation, caspase activation and ultimately cell death. Several crystal structures of the ExtraCellular Domain from Death Receptor 5 (DR5-ECD) have been reported in complex with the TRAIL ligand or anti-DR5 antibodies, but none for the isolated protein. In order to fill this gap and to perform binding experiments with TRAIL peptidomimetics, we have produced isotopically labelled DR5-ECD and started a conformational analysis by using high-field 3D NMR spectroscopy. Herein, we present the first resonance assignment of a TRAIL receptor in solution and the determination of its secondary structure from NMR chemical shifts.     

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-kappaB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-kappaB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a transducer for proinflammatory and angiogenic signals in human brain tumors.