Factor Receptor Superfamily Member Research Articles

Effects of denosumab treatment on the expression of receptor activator of nuclear kappa-B ligand (RANKL) and TNF-receptor TNFRSF9 after total hip arthroplasty—results from a randomized placebo-controlled clinical trial

SummaryWe investigated whether the drug denosumab modulates the inflammatory response after total hip arthroplasty in a randomized controlled trial. Significantly increased expression of RANKL was found in patients treated with denosumab. This could provide an explanation for the rebound effect with rapid loss of BMD seen after discontinuation of denosumab treatment.PurposeTo evaluate whether denosumab, a human monoclonal antibody directed against receptor activator of nuclear factor kappa-B ligand (RANKL), modulates the inflammatory response after cementless total hip arthroplasty (THA) in patients with osteoarthritis of the hip.MethodsSixty-four patients operated with cementless THA were randomized to two doses of 60-mg denosumab or placebo 1–3 days and 6 months postoperatively. Serum samples were analyzed by a multiplex extension assay detecting 92 inflammation-related proteins. Bone turnover markers were assessed. Proteins were analyzed using linear mixed effect models. Validation of conspicuous findings was performed with ELISA.ResultsTwo proteins were significantly affected by denosumab treatment: RANKL and tumor necrosis factor receptor super family member 9 (TNFRSF9). Serum levels of RANKL were more than twice as high in the denosumab than in the placebo group 3 months after surgery (ratio 2.10, p<0.001). Six and 12 months after surgery, the expression of RANKL was still elevated in the denosumab-treated group (ratios 1.50, p < 0.001; 1.47, p =0.002). The expression of TNFRSF9 was lower in the denosumab group at 3 months (ratio 0.68, p<0.001). In the denosumab group, concentrations of bone turnover markers were substantially reduced after 3 months, remained suppressed after 6 and 12 months, but increased above baseline at 24 months after surgery.ConclusionTwo subcutaneous denosumab injections 6 months apart increase RANKL and depress TNFRSF9 after THA. This provides a possible explanation for the rebound effect on bone turnover markers as well as bone mineral density (BMD) upon withdrawal of denosumab. None of the other measured markers of inflammation was influenced by denosumab treatment.

Differentially expressed inflammatory proteins in acute gouty arthritis based on protein chip

To detect the differentially expressed inflammatory proteins in acute gouty arthritis (AGA) with protein chip. The Raybiotech cytokine antibody chip was used to screen the proteomic expression in serum samples of 10 AGA patients and 10 healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to determine the biological function annotation of differentially expressed proteins and the enrichment of signal pathways. ELISA method was used to verify the differential protein expression in 60 AGA patients and 60 healthy subjects. The ROC curve was employed to evaluate the diagnostic value of differential proteins in AGA patients. According to|log2FC|>log2 1.2 and corrected P<0.01, 4 most differentially expressed proteins in AGA patients were identified, including tumor necrosis factor receptor super family members Ⅱ (TNF RⅡ), macrophage inflammatory protein 1β (MIP-1β), interleukin-8 (IL-8), and granulocyte-macrophage colony stimulating factor (GM-CSF). GO and KEGG enrichment analysis showed that the differentially expressed proteins were related to inflammation, metabolism and cytokine pathways. The ELISA results showed that serum levels of differentially expressed proteins were significantly different between AGA patients and healthy subjects(all P<0.01). ROC curve analysis showed that the areas under the curve (AUCs) of GM-CSF, IL-8, MIP-1β and TNF RⅡ for predicting AGA were 0.657 (95% CI: 0.560-0.760, sensitivity: 68.33%, specificity: 50.00%), 0.994 (95% CI: 0.980-1.000, sensitivity: 100.00%, specificity: 61.67%), 0.980 (95% CI: 0.712-0.985, sensitivity: 95.00%, specificity: 98.33%) and 0.965 (95% CI: 0.928-1.000, sensitivity: 100.00%, specificity: 10.00%), respectively. Proteomics can be applied to identify the biomarkers of AGA, which may be used for risk prediction and diagnosis of AGA patients.

Abstract 563: A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking

Abstract The development of agonistic antibodies that activate T-cell co-stimulatory pathways represents a therapeutic strategy with significant clinical potential. However, challenges remain for the translation from in vitro efficacy to clinical success. OX40 and other tumor necrosis factor receptor (TNFR) superfamily members are notorious for requiring high-order receptor clustering in order to achieve full activity. For monoclonal antibodies, this high-order clustering is generally achieved through secondary cross-linking strategies. In vivo, this secondary cross-linking is often supplied through internal immune effector cells via Fc engagement. Bispecific and biparatopic antibodies represent an emerging class of drug molecules that enable unique mechanisms of action relative to their monoclonal counterparts. Here, we describe the use of our bispecific platform for the generation of large panels of biparatopic antibodies which enabled high-throughput screening for the discovery of an array of OX40 agonistic molecules. We have optimized these multivalent antibodies that exceed the potency of the OX40 ligand in NFkB activation without the need for secondary cross-linking. These agonist antibodies have additionally been characterized using primary T cell assays to monitor the kinetics of growth proliferation and cytokine secretion, outperforming cross-linked antibodies currently being tested in clinical trials. In co-culture systems, these agonist antibodies were effective in inhibiting immuno-suppressive properties of Tregs. Our lead OX40 agonist antibody has been optimized for activity and developability and has entered stable cell line development to further support pre-clinical activities. Citation Format: Bonnie Hammer, Mandar Bawadekar, Matthew Bissen, John Painter, Lauren Lehmann, Francis Qufei Li, Lucas Bailey, Bryan Glaser, Roland Green. A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 563.

Chronic retroviral infection of mice promotes tumor development, but CD137 agonist therapy restores effective tumor immune surveillance.

T cell responses are crucial for anti-tumor immunity. In chronic viral infections, anti-tumor T cell responses can be compromised due to various immunological mechanisms, including T cell exhaustion. To study mechanisms of anti-tumor immunity during a chronic viral infection, we made use of the well-established Friend virus (FV) mouse model. Chronically FV-infected mice are impaired in their ability to reject FBL-3 cells-a virus-induced tumor cell line of C57BL/6 origin. Here we aimed to explore therapeutic strategies to overcome the influence of T cell exhaustion during chronic viral infection, and reactivate effector CD8+ and CD4+ T cells to eliminate tumor cells. For T cell stimulation, agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily members CD137 and CD134 were used, because they were reported to augment the cytotoxic program of T cells. αCD137 agonistic therapy, but not αCD134 agonistic therapy, resulted in FBL-3 tumor elimination in chronically FV-infected mice. CD137 stimulation significantly enhanced the cytotoxic activity of both CD4+ and CD8+ T cells, which were both required for efficient tumor control. Our study suggests that agonistic antibodies to CD137 can efficiently enhance anti-tumor immunity even in the setting of chronic viral infection, which might have promising therapeutic applications.

Mutations in osteoprotegerin account for the CCAL1 locus in calcium pyrophosphate deposition disease

Mutations on chromosomes 5p (CCAL2) and 8q (CCAL1) have been linked to familial forms of calcium pyrophosphate deposition disease (CPDD). Mutations in the ANKH gene account for CCAL2, but the identity of CCAL1 has been elusive. Recently, a single Dutch kindred with a mutation in the Tumor Necrosis Factor Receptor Super Family member 11B (TNFRSF11B) gene coding for osteoprotegerin (OPG) was described as a gain-of-function mutation. Affected family members had premature generalized osteoarthritis (PGOA) and CPDD. As the TNFRSF11B gene is on 8q, we sought additional evidence that TNFRSF11B was CCAL1, and investigated potential disease mechanisms. DNA from two novel PGOA/CPDD families was screened for sequence variants in the TNFRSF11B gene. Mutations were verified by genotype analysis of affected and unaffected family members. We also investigated effects of normal and mutant OPG on regulators of CPP crystal formation in porcine cartilage. The identical TNFRSF11B mutation described in the Dutch family was present in two novel PGOA/CPDD families. ANKH was normal in affected patient fibroblasts. Exogenous OPG did not alter ANKH mRNA or protein levels, affect translocation of ANKH to the membrane, nor increase [pyrophosphate (PPi)] or other key regulators of CPDD. We have firmly established the identity of CCAL1 as TNFRSF11B (OPG). Our findings suggest that this mutation produces disease in an ANKH-independent manner via novel mechanisms not primarily targeting cartilage. This work rationalizes further investigation of OPG pathway components as potential druggable targets for CPDD.

Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib.

Recently tumor necrosis factor receptor super family member 18 (TNFRSF18, also called GITR) has been identified as a novel tumor suppressor gene in Multiple Myeloma (MM), undergoing aberrant DNA methylation-mediated gene expression silencing. Furthermore, the expression of GITR blocks canonical NF-κB activation in MM cells in response to TNFα. Bortezomib, a proteasome inhibitor, can induce NF-κB activation, which may significantly influence the drug response in MM patients. In this study, we aim to elucidate if GITR status is associated with response to Bortezomib in MM cells through regulating GITR mediated NF-κB blockade. We found that GITR was significantly downregulated in MM patients and cell lines. Overexpression of GITR inhibited non-canonical NF-κB activation induced by TNFα. Moreover, NF-κB inhibitor induced apoptosis in GITR-deficient MM cells in response to TNFα. In addition, overexpression of GITR could inhibit Bortezomib-induced NF-κB activation and enhance the cytotoxicity of Bortezomib in GITR-deficient MM cell line (MM1.S). In contrast, knockdown of GITR attenuated the cytotoxic effect of Bortezomib on GITR proficient MM (RPMI) cell line and increased NF-κB activation. Finally, overexpression of GITR enhanced the sensitivity to Bortezomib in co-culture with bone marrow stromal cells and significantly reduced the tumor growth in MM1.S xenograft mice. In conclusion, we demonstrated that GITR expression can enhance the sensitivity to Bortezomib by inhibiting Bortezomib-induced NF-κB activation.

Abstract 497: Engagement of activatory Fc γ receptors by immunoregulatory receptor-targeting antibodies mediates antitumor activity.

Abstract Inhibitory FcγRIIB receptors are required for the therapeutic effects of agonistic antibodies targeting several tumor necrosis factor receptor (TNFR) superfamily members, including CD40, DR5, and Fas. However, it remains unclear how general this requirement is and whether activating FcγRs may be required for antibodies targeting other immunoregulatory receptors. Here we have explored the involvement of FcγRs for the antitumor effects of antibodies targeted to costimulatory TNFRs expressed on T cells. Surprisingly, FcγRIIB was completely dispensable for antibody-mediated anti-tumor effects. In contrast, activating FcγRs were required for maximal in vivo activity. A similar dependence on activating FcγRs was observed for antibodies targeted to the T cell surface antigen CTLA-4. Important changes in the tumor microenvironment and draining lymph nodes correlated with the in vivo efficacy of these antibodies. Our findings uncover a previously unknown requirement for activating FcγRs in the anti-tumor effects of immunomodulatory antibodies and raise important questions about the role of receptor signaling for therapeutic activity. A greater understanding of this activatory FcγR-based mechanism may improve the design and activity of immune-modulatory antibodies in clinical development, and those already approved for the clinic. Citation Format: Yannick Bulliard, Rose Jolicoeur, Maurice Windman, Sarah Rue, Deborah Knee, Nick Wilson, Glenn Dranoff, Jennifer Brogdon. Engagement of activatory Fc γ receptors by immunoregulatory receptor-targeting antibodies mediates antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2013-497

Association between CD40 promoter polymorphism (rs1800686, −508 C/T) and ischemic stroke with hypertension in a Korean population

Although glutamate toxicity is important in stroke injury, the immune and inflammatory systems are also related to stroke pathogenesis. Tumor necrosis factor (TNF) receptor superfamily member 5 (CD40) plays a crucial role in mediating a broad variety of immune and inflammatory responses. To investigate whether CD40 polymorphisms are associated with the development of ischemic stroke, two promoter single nucleotide polymorphisms (SNPs) of the CD40 gene (rs1800686, −508A/G; rs752118, −173C/T) were analyzed in 121 patients with ischemic stroke (IS) and 291 control subjects. All stroke patients were classified into clinical subgroups according to their blood pressure levels (hypertension, present and absent), fasting plasma glucose (diabetes mellitus, present and absent), and lipids (dyslipidemia, present and absent). SNPStats and SNPAnalyzer Pro software were used to obtain odds ratios (ORs), 95% confidence intervals (CIs), and Pvalues. Multiple logistic regression models (codominant1, codominant2, dominant, recessive, overdominant, and log-additive) were used to analyze the genetic data. The two tested CD40 promoter SNPs (rs1800686 and rs752118) were not associated with the development of IS, but the GT haplotype of these two SNPs was associated with the development of IS (P= 0.0148). The rs1800686 SNP frequency differed between IS patients without hypertension and those with hypertension (P=0.017, OR=0.17, 95% CI=0.04–0.72 in the codominant2 model; P=0.023, OR=0.37, 95% CI=0.15–0.90 in the dominant model; P=0.0085, OR=0.42, 95% CI=0.21–0.81 in the log-additive model; P=0.011, OR=0.47, 95% CI=0.27–0.85 in allele frequencies). The A allele frequency of rs1800686 was lower in IS patients with hypertension (27.2%) than in IS patients with hypertension (44.3%), while the G allele frequency of rs1800686 was higher in IS patients with hypertension (72.8%) than in IS patients with hypertension (55.7%). These results suggest that the GT haplotype of rs1800686 and rs752118 in the CD40 gene may be associated with the development of IS, and rs1800686 may contribute to IS in Korean patients with hypertension.

Dcr3 inhibit p53-dependent apoptosis in γ-irradiated lung cancer cells

Purpose: To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines.Materials and methods: mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay.Results: From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P = 4.38 × 10−7) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway.Conclusion: Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.

The anti-Fas antibody M-20 cross-reacts with intracellular proteins: implications for false-positive detection of Fas

Fas (CD95, APO-1) is prototypical Tumor Necrosis FactorReceptor (TNFR) superfamily member and its role in influ-encing cell death and survival has been intensively studiedin multiple contexts including inflammation, developmentand degeneration (reviewed in [1]). Fas expression is wellcharacterized in the immune system but its constitutive andregulated expression in other tissues is less well understood.M-20(SantaCruzBiotechnology,catalogsc-716)isanaffin-ity purified polyclonal rabbit anti-Fas antibody raised to apeptide at the carboxyl terminus of mouse Fas. According tothe manufacturer, M-20 is suitable for immunoblotting, im-

Autoimmune lymphoproliferative syndrome: etiology, diagnosis, and management.

Autoimmune lymphoproliferative syndrome (ALPS) is a childhood disorder characterized by chronic, nonmalignant lymphoproliferation and autoimmunity, most commonly involving cells of hematopoietic origin. Mutations of the tumor necrosis factor receptor super family member 6 (TNFRSF6) gene, coding for the apoptosis-inducing protein Fas (Apo-1, CD95) are involved in the physiopathology of the syndrome, although the complete mechanism by which the syndrome is caused has not yet been unraveled. Although the syndrome has a benign nature, life-threatening complications can demand treatment. Treatment schedules, including corticosteroids, low doses of chemotherapy, granulocyte colony stimulating factor, or splenectomy, have varying results. Treatment with the antimalarial drug pyrimethamine/sulfadoxine (25/500mg per tablet) seems to be a new, well tolerated, and efficient approach, although larger studies will have to demonstrate the true value of this drug in patients with ALPS.

CD40 and B Chronic Lymphocytic Leukemia Cell Response To Fludarabine: The Influence of NF-kB/Rel Transcription Factors On Chemotherapy-Induced Apoptosis

The levels of tumour necrosis factor receptor (TNF-R) superfamily members can be altered in lymphoid leukemias, indicating a possible role of such molecules in the biology of these neoplasias. In B chronic lymphocytic leukemia cells, the CD40/CD40L system has been shown to be effective in inhibiting the apoptotic response to fludarabine. The modulation of apoptosis relied on the CD40- induced activity of NF-B/Rel transcription factors. The anti- apoptotic effect of CD40 was abolished using a phosphorothioate B decoy oligodeoxynucleotide. These findings illustrate an example of the biological activity of TNF-R- like molecules in leukemias. They also show the influence of NF-B/Rel activity on leukemic cell response to apoptogenic agents.