anti-DR5 mAb Research Articles

Combination of multivalent DR5 receptor clustering agonists and histone deacetylase inhibitors for treatment of colon cancer

Death Receptor 5 (DR5) targeted therapies offer significant promise due to their pivotal role in mediating the extrinsic pathway of apoptosis. Despite DR5 overexpression in various malignancies and the potential of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), clinical applications of anti-DR5 monoclonal antibodies (mAbs) have been hampered by suboptimal outcomes potentially due to lack of receptor clustering.To address the limitation, we developed N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based conjugates integrating multiple copies of DR5-targeting peptide (cyclic WDCLDNRIGRRQCVKL; cDR5) to enhance receptor clustering and apoptosis. Three conjugates with variable number of cDR5 were prepared and denoted as PH-cDR5 (high valence), PM-cDR5 (medium valence) and PL-cDR5 (low valence). Our studies in TRAIL-sensitive and resistant cancer cell lines demonstrated that the HPMA copolymer-peptide conjugates (P-cDR5) significantly improved DR5 receptor clustering and induced apoptosis effectively. In TRAIL-sensitive colon cancer cells (COLO205, HCT-116), P-cDR5 showed efficacy comparable to anti-DR5 mAb Drozitumab (DRO), but P-cDR5 outperformed DRO in TRAIL-resistant cells (HT-29), highlighting the importance of efficient receptor clustering. In COLO205 cells PM-cDR5 exhibited an IC50 of 94 pM, while PH-cDR5 had an even lower IC50 of 15 pM (based on cDR5 equivalent concentration), indicating enhanced potency of the multivalent HPMA copolymer-based system with a flexible polymer backbone in comparison with the IC50 for TRAIL at 0.12 nM. Combining P-cDR5 with valproic acid, a histone deacetylase inhibitor, resulted in further enhancement of apoptosis inducing efficacy, along with destabilizing mitochondrial membranes and increased sensitivity of TRAIL-resistant cells. These findings suggest that attaching multiple cDR5 peptides to a flexible water-soluble polymer carrier not only overcomes the limitations of previous designs but also offers a promising avenue for treating resistant cancers, pointing toward the need for further preclinical exploration and validation of this innovative strategy.

Abstract 3493: New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity

Abstract Death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2 or Apo2) are TNF receptor superfamily members that are receptors for TRAIL (Apo2 ligand), an immunomodulatory cytokine. Binding of TRAIL to DR4 or DR5 can activate the extrinsic apoptotic pathway selectively in tumor cells. Agonist antibodies to death receptors DR4 and DR5 thus have potential for treatment of cancer and may be better than derivatives of TRAIL itself because of their superior pharmacokinetics and receptor selectivity. However, despite dramatic effects in vitro and in preclinical xenograft models, several agonist antibodies to DR4 and especially DR5 have not provided significant therapeutic benefit in clinical trials. While resistance mechanisms in cancer cells are undoubtedly one reason, another factor may be inadequate potency of the antibodies tested. We therefore used intense immunization and screening protocols to develop very potent anti-DR4 and anti-DR5 monoclonal antibodies (mAbs), which were humanized to make the mAbs denoted HuD114 and HuG4.2 respectively. These mAbs were substantially more effective at killing tumor cells in vitro than the mAbs that have been tested in clinical trials. In addition, we introduced one or two mutations into the Fc (constant) regions of these and other potent anti-DR4 and anti-DR5 mAbs in order to increase affinity for the Fc gamma receptor IIb. Cross-linking, as provided by binding of anti-DR4 and anti-DR5 mAbs to Fc gamma receptors on immune cells, is required for effective transmission of an apoptotic signal through the death receptors. Accordingly, introduction of these mutations greatly increased the ability of the mAbs to kill tumor cells in vitro in the presence of human peripheral blood mononuclear cells, and to inhibit the growth of tumor xenografts in mouse models, with two mutations generally more effective than a single mutation. As a complementary approach to increase cross-linking, we also developed bispecific antibodies containing two binding domains from an anti-DR4 mAb and two binding domains from an anti-DR5 mAb. These bispecific mAbs were made in the IgG-like Bs(scFv)4-IgG format consisting of a homodimer of two monomers, each monomer having a single chain Fv from an anti-DR4 mAb linked to a heavy chain constant region, and a single chain Fv from an anti-DR5 mAb linked to a light chain constant region. Such a bispecific mAb was more effective at killing tumor cells than an anti-DR4 or anti-DR5 mAb alone, or even a mixture of anti-DR4 and anti-DR5 mAbs. Based on their potency in vitro and in animal models, we believe that HuD114 and HuG4.2, enhanced by the Fc mutations and/or in bispecific form, have the potential for greater clinical efficacy than previously tested anti-DR4 and anti-DR5 mAbs and thus merit further investigation. Citation Format: Lihong Wang, Yi Ding, Hangil Park, April Zhang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3493.

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression.

Anti-DR5 mAb inhibits proliferation of human fibroblast-like synovial cells and reduces their cytokine secretion in vitro.

BackgroundWe have previously reported that anti-death receptor 5 (DR5) monoclonal antibody (mAb) is therapeutically effective in the treatment of rheumatoid arthritis (RA) in a collagen-induced arthritis rat model. However, the molecular mechanism and the effect of anti-DR5 mAb on proapoptotic genes and cytokine secretion in the human fibroblast-like synovial cells (FLS) requires further clarification. This study may provide new evidence for the application of anti-DR5 mAb as a treatment for RA.MethodsHuman FLS were isolated from patients with RA and were treated with anti-DR5 mAb. An MTT assay and flow cytometry were used to detect the induction of apoptosis in vitro. Cytokine secretion by the FLS was detected using the enzyme-linked immunosorbent assay. The mRNA expression was assessed by reverse transcription polymerase chain reaction, and the protein expression was analyzed by Western blot. The apoptotic pathway was investigated further using a caspase inhibition assay.ResultsAnti-DR5 mAb-induced apoptosis in human RA FLS in vitro. The protein expressions of caspase-8, -3, and -9 were decreased in human anti-DR5 mAb-treated FLS in a dose-dependent manner through exposure to a caspase inhibitor, indicating that anti-DR5 mAb induction of apoptosis is through the caspase pathway. Decreased levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were detected after treatment with anti-DR5 mAb in vitro.ConclusionAnti-DR5 mAb may induce apoptosis in human FLS through the caspase pathway and through decreased secretions of TNF-α and IFN-γ.

Abstract 2464: DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity

Abstract Introduction: Death Receptor 5 (DR5) belongs to the tumor necrosis factor receptor superfamily. The target and its associated signaling pathway are preferentially active on cancer cells. DR5 elicits pro-apoptotic signal through receptor oligomerization upon binding of TRAIL or an agonistic mAb. Chemotherapy and radiation also can synergize with the DR5 pathway. Various therapeutics targeting DR5 have been generated but clinical outcomes have been generally disappointing. Here we describe optimization of the agonistic activity of anti-DR5 mAbs by enabling multivalent target engagement via engineering into DARTs; covalently-linked Fv-based diabodies optimized for manufacturability and stability (1) whose modular nature facilitates tailoring avidity and PK properties to the specific needs of the application. Methods: Anti-DR5 mAbs were selected from a panel of mAbs generated from cancer cell immunizations and characterized for binding properties. DARTs with varying anti-DR5 specificities, valency and incorporation of either a wild type IgG1 Fc or an Fc mutated to eliminate FcγR binding, were expressed in CHO cells and purified to homogeneity. DR5 DARTs and mAbs were characterized across a panel of cell lines including those derived from colorectal, lung, pancreatic, breast and prostate cancer and by IHC on normal and tumor tissue specimens. Results: mAbs from whole cancer cell immunizations displaying differential expression on both normal and cancer tissues were subjected to antigen identification and identified a subset with reactivity to DR5. Binding analyses revealed DR5 mAbs recognizing non-overlapping epitopes that do not block the TRAIL-DR5 interaction. Upon secondary cross-linking or when combined, the DR5 mAbs induced apoptosis across multiple cancer cell lines. Further potency enhancement independent of secondary cross-linking was obtained by the engineered multivalent DR5-targeting DARTs. Dose-dependent growth inhibition assay demonstrated that the DR5-targeting DARTs delivered significantly greater potency than TRAIL (≥100-fold) across a broad range of cancer cell lines. Furthermore, the DR5-targeting DARTs maintained the exquisite tumor/normal differential reactivity displayed by the parental DR5 mAbs from which they were derived. Combination of an HDAC inhibitor with DR5-targeting DARTs revealed maintenance of synergistic activity and potential to overcome pathway resistance. Conclusion: Whole cancer cell immunizations yielded DR5 mAb candidates with desirable binding and functional properties. Through incorporation of the anti-DR5 mAb specificities into multivalent DART molecules, we have generated a new class of therapeutics that may overcome the limitations of existing DR5-based therapeutics. The data support the use of DR5-targeting DARTs to target this apoptotic pathway in multiple cancer cells. Reference: (1) Johnson et al. 2010. J Mol Bio. 399:436-49 Citation Format: Jonathan C. Li, Kalpana Shah, Jill Rillema, Francine Chen, Doug Smith, Steve Burke, Valentina Ciccarone, Sergey Gorlatov, Ralph Alderson, James Tamura, Ezio Bonvini, Syd Johnson, Paul A. Moore. DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2015-2464

Abstract 2271: Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists

Abstract Metastatic renal cell carcinoma (RCC) is incurable. IFNα or IL-2 immunotherapy has been used for RCC for more than 20 years, but response rates remain low and these cytokines can generate toxic. More recently, administration of multikinase inhibitors or anti-VEGF receptor mAb has become popular, but these agents are largely palliative and tumor regression is common. The shortcomings in current therapeutic options for patients with metastatic RCC provide the rationale for the development of novel treatment protocols. Triptolide, a diterpene triepoxide from the medicinal herb Tripterygium wilfordii, can suppress cell growth and induce apoptosis in a range of human tumor cells. Further, triptolide also sensitizes tumor cells to TNF-related apoptosis-inducing ligand (TRAIL). The objective of this study was to determine the effectiveness of combining triptolide with TRAIL receptor agonists against RCC. In vitro, the combination of triptolide and recombinant human TRAIL protein decreased cell viability in the human RCC cell line ACHN cells compared with either treatment alone. This decrease in viability correlated with increased induction of apoptosis (positive Annexin V staining) and caspase activation. The triptolide-induced sensitization was accompanied by decreased HSP70, HSP27, and HSF1 expression, as well as increased surface expression of TRAIL-R2. Similar in vitro treatment of the mouse renal cell adenocarcinoma cell line, Renca, demonstrated that triptolide enhanced the killing of these cells by membrane-bound TRAIL or agonistic anti-DR5 mAb. In vivo treatment of mice bearing orthotopic and metastatic Renca tumors showed that combination therapy consisting of minnelide (a water soluble version of triptolide) and agonistic anti-DR5 mAb resulted in significantly decreased tumor burden and increased animal survival compared to either therapy alone. Collectively, our data suggest that triptolide sensitizes RCC cells to TRAIL-induced apoptosis, which is accompanied by decreased heat shock protein expression and altered TRAIL receptor expression. These data also suggest that the combination of TRAIL receptor agonists and triptolide may prove to be an effective treatment for metastatic RCC, especially at doses that show suboptimal tumoricidal activity when used individually. Citation Format: Thomas S. Griffith, Erik L. Brincks, Tamara A. Kucaba, Britnie R. James, Veena Sangwan, Sulagna Banerjee, Ashok Saluja. Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2014-2271

Histone Deacetylase Inhibitor Sensitizes Apoptosis-Resistant Melanomas to Cytotoxic Human T Lymphocytes through Regulation of TRAIL/DR5 Pathway

Modern immune therapies (PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer) have remarkably improved the response rates of metastatic melanoma. These modalities rely on the killing potential of CTL as proximal mediator of antimelanoma responses. Mechanisms of tumor resistance to and the predominant cytotoxic pathway(s) used by melanoma-reactive CTL are important outcome determinants. We hypothesized that downmodulation of death receptors (DRs) in addition to aberrant apoptotic signaling might confer resistance to death signals delivered by CTL. To test these two hypotheses, we used an in vitro model of MART CTL-resistant melanoma sublines. TCR-transgenic and patient-derived CTLs used the TRAIL cytotoxic pathway through DR5. Furthermore, recombinant human TRAIL and drozitumab (anti-DR5 agonistic mAb) were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. CTL resistance was due to DR5 downregulation and an inverted ratio of pro- to antiapoptotic molecules, both of which were reversed by the histone deacetylase inhibitor suberoylanilide hydroxanic acid. Apoptosis negative (c-IAP-2 and Bcl-xL) and positive (DR5) regulators were potential incriminators partly regulating CTL sensitivity. These preclinical findings suggest that exposure to this chromatin remodeling drug of immune-resistant melanomas can skew toward an intracellular proapoptotic milieu, increase DR expression, and overcome acquired immune resistance.

Additive effect of sirolimus and anti‐death receptor 5 agonistic antibody against hepatocellular carcinoma

Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC. We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb+sirolimus. Anti-DR5 mAb (200 and 300μg/day) induced liver dysfunction with partial necrosis of the liver, but 100μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups. Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

Retraction Note: Induction of tumor cell apoptosis via Fas/DR5

The apoptosis inducing effects on tumor cell lines MGC803, BEL7402 and HL60 by Fas ligand and anti-human DR5 monoclonal antibodies (anti-DR5 mAb) and the underlying mechanism was studied. Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/anti-DR5 mAb on tumor cell lines was measured by MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. Flow cytometry was employed to analyze the mode of cell death. The mRNA expression of DR5 in MGC803 and BEL7402 cells after giving anti-DR5 mAb was up-regulated compared with control group, while it was down-regulated in HL60 cells in the same condition. The mRNA expression of Fas in HL60 was higher after giving FasL compared with control group, while it was lower in MGC803 and BEL7402. MGC803 and BEL7402 were sensitive to anti-DR5 mAb but partially to FasL, and HL60 was sensitive to FasL but less sensitive to anti-DR5 mAb. Apoptosis induced by Fas ligand and anti-DR5 mAb vary among tumor cell lines. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, which was presented as the release of caspase-8 and Bcl-2.

Induction of tumor cell apoptosis via Fas/DR5

The apoptosis inducing effects on tumor cell lines MGC803, BEL7402 and HL60 by Fas ligand and anti-human DR5 monoclonal antibodies (anti-DR5 mAb) and the underlying mechanism was studied. Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/anti-DR5 mAb on tumor cell lines was measured by MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. Flow cytometry was employed to analyze the mode of cell death. The mRNA expression of DR5 in MGC803 and BEL7402 cells after giving anti-DR5 mAb was up-regulated compared with control group, while it was down-regulated in HL60 cells in the same condition. The mRNA expression of Fas in HL60 was higher after giving FasL compared with control group, while it was lower in MGC803 and BEL7402. MGC803 and BEL7402 were sensitive to anti-DR5 mAb but partially to FasL, and HL60 was sensitive to FasL but less sensitive to anti-DR5 mAb. Apoptosis induced by Fas ligand and anti-DR5 mAb vary among tumor cell lines. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, which was presented as the release of caspase-8 and Bcl-2.

TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice

Apoptotic death of alveolar macrophages observed during lung infection with Streptococcus pneumoniae is thought to limit overwhelming lung inflammation in response to bacterial challenge. However, the underlying apoptotic death mechanism has not been defined. Here, we examined the role of the TNF superfamily member TNF-related apoptosis-inducing ligand (TRAIL) in S. pneumoniae-induced macrophage apoptosis, and investigated the potential benefit of TRAIL-based therapy during pneumococcal pneumonia in mice. Compared with WT mice, Trail(-/-) mice demonstrated significantly decreased lung bacterial clearance and survival in response to S. pneumoniae, which was accompanied by significantly reduced apoptosis and caspase 3 cleavage but rather increased necrosis in alveolar macrophages. In WT mice, neutrophils were identified as a major source of intraalveolar released TRAIL, and their depletion led to a shift from apoptosis toward necrosis as the dominant mechanism of alveolar macrophage cell death in pneumococcal pneumonia. Therapeutic application of TRAIL or agonistic anti-DR5 mAb (MD5-1) dramatically improved survival of S. pneumoniae-infected WT mice. Most importantly, neutropenic mice lacking neutrophil-derived TRAIL were protected from lethal pneumonia by MD5-1 therapy. We have identified a previously unrecognized mechanism by which neutrophil-derived TRAIL induces apoptosis of DR5-expressing macrophages, thus promoting early bacterial killing in pneumococcal pneumonia. TRAIL-based therapy in neutropenic hosts may represent a novel antibacterial treatment option.

Anti-DR5 mAb Ameliorate Adjuvant Arthritis Rats Through Inducing Synovial Cells Apoptosis

Study the therapeutic effects and immunoregulatory mechanisms of anti-DR5 mAb on adjuvant arthritis (AA) rats. AA rats induced by CFA, were treated with anti-DR5 mAb through mainline administration. Effect on the synovial membranes of the tissues was detected by H&E staining. Flow cytometry and MTT assay were used for detecting the induced apoptosis in an in vitro system and TUNEL assay was used for analysis in an in vivo system. The involvement of the apoptotic pathway was further proved by a caspase inhibition assay. Anti-DR5 mAb could induce synovial cell apoptosis in an in vitro system, which was related with the mRNA expression of DR5 on the cell surface. The mRNA expressions of c-myc and bcl-2 were decreased in synovial cells and those of p21, p53, and bax were increased. The protein expressions of caspase-8/3/9, RANKL, JNK2, and c-Jun were raised and that of bcl-2 was decreased. When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb, it showed a dose-dependence inhibition effect, indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway. This study shows that anti-DR5 mAb can ameliorate arthritic symptoms. The mechanisms of the treatment are related to the increase in synovial cell apoptosis by regulating the mRNA expression of DR5 and apoptosis-related genes, prolonging the duration of the cell cycle by modulation of the mRNA expression of cell cycle-related genes, and the protein expression of the molecules in the caspase pathway and RANKL, JNK2, and c-Jun.

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

Antibodies targeted to TRAIL receptor-2 and ErbB-2 synergize in vivo and induce an antitumor immune response

Despite the development of human epidermal growth factor receptor-2 (ErbB-2/HER2)-targeted therapies, there remains an unmet medical need for breast cancer patients with ErbB-2 overexpression. We investigated the therapeutic activity of an agonist mAb to mouse tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 (DR5) against ErbB2-driven breast cancer. Established tumors in BALB/c transgenic mice expressing a constitutively active ErbB-2/neuT were treated with anti-DR5 mAb and/or anti-ErbB-2 mAb and monitored for tumor progression. Treatment with anti-DR5 or anti-ErbB2 mAb as single agents significantly delayed tumor growth, although all tumors eventually progressed. Remarkably, treatment with a combination of anti-DR5 and anti-ErbB-2 mAbs induced complete response in a majority of mice. In vivo blockade of CD11b(+) cells, but not natural killer cell depletion, significantly abrogated the early antitumor response. Notably, depletion of CD8(+) T cells provoked primary and secondary tumor relapse, revealing the induction of antitumor immunity by the combination treatment. Combined therapy with anti-DR5 and anti-ErbB-2 mAbs further significantly suppressed the growth of advanced spontaneous tumors in ErbB-2/neuT transgenic mice, even when treatment was delayed until tumors were palpable. We thus demonstrated that the combination of anti-DR5 and anti-ErbB2 mAbs might be an effective form of treatment for ErbB-2-overexpressing breast cancer.

Combination antibody‐based cancer immunotherapy

It is now commonly recognized that tumor-targeting monoclonal antibodies (mAb) demonstrate significant therapeutic effects in cancer patients. Multiple effector mechanisms are involved in the primary response; however, the induction of tumor-specific immunity has sometimes been a desired outcome and in some cases has contributed to the success of mAb-based therapy. Initiating the antitumor immune response by creating a tumor antigen supply from dying (apoptotic) tumor cells is a goal of tumor-targeting mAb therapy, and ideally a sufficiently immunogenic tumor cell death might provide a platform on which combination immunotherapies can be based. In this review, the authors discuss the possible utility and pitfalls of using such a therapy, which combines tumor cell death with immune activation therapy. The ideas are largely based on the observation that three-mAb (anti-DR5 mAb, anti-CD40 mAb, and anti-CD137 mAb [trimAb]) therapy has been shown to be very effective in a number of mouse experimental tumor models. The authors believe that this rational activation and inhibition of key points in the immune response can be an effective strategy to apply in human cancer patients.

Induction of tumor-specific T cell immunity by anti-DR5 antibody therapy.

Because tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells and plays a critical role in tumor surveillance, its receptor is an attractive target for antibody-mediated tumor therapy. Here we report that a monoclonal antibody (mAb) against the mouse TRAIL receptor, DR5, exhibited potent antitumor effects against TRAIL-sensitive tumor cells in vivo by recruiting Fc receptor–expressing innate immune cells, with no apparent systemic toxicity. Administration of the agonistic anti-DR5 mAb also significantly inhibited experimental and spontaneous tumor metastases. Notably, the anti-DR5 mAb-mediated tumor rejection by innate immune cells efficiently evoked tumor-specific T cell immunity that could also eradicate TRAIL-resistant variants. These results suggested that the antibody-based therapy targeting DR5 is an efficient strategy not only to eliminate TRAIL-sensitive tumor cells, but also to induce tumor-specific T cell memory that affords a long-term protection from tumor recurrence.