TNFRSF10B Antibody

What is TNFRSF10B and what is its role in cell signaling?

TNFRSF10B, also known as TRAIL R2, DR5, and TRICK 2, is a type 1 membrane protein belonging to the TNF receptor superfamily (TNFRSF). It functions as a receptor for TRAIL (APO2 ligand) and plays a critical role in apoptosis signaling pathways. The TNFRSF10B cDNA encodes a 440 amino acid residue precursor protein containing extracellular cysteine-rich domains, a transmembrane domain, and a cytoplasmic death domain . Among the TNF receptor family proteins, TNFRSF10B is most closely related to TRAIL R1/DR4, sharing approximately 55% amino acid sequence identity .

The primary signaling function of TNFRSF10B involves the induction of apoptosis. When trimeric TRAIL binds to TNFRSF10B on the cell surface, it induces oligomerization of the receptor, which is necessary for initiating downstream apoptotic signaling. This receptor oligomerization facilitates the formation of the death-inducing signaling complex (DISC), leading to the activation of initiator caspases and subsequent executioner caspases, ultimately resulting in programmed cell death.

Beyond its role in apoptosis, TNFRSF10B signaling also intersects with other pathways, including NF-κB activation, which has been implicated in myeloid cell maturation, as demonstrated in studies showing that "TRAIL and TNF-alpha promote the NF-kappaB-dependent maturation of normal and leukemic myeloid cells" . This highlights the context-dependent nature of TNFRSF10B signaling outcomes.

What are the common applications of TNFRSF10B antibodies in research?

TNFRSF10B antibodies serve multiple critical functions in biomedical research, with applications spanning protein detection, functional studies, and therapeutic development. The most commonly employed applications include:

Western Blot Analysis: TNFRSF10B antibodies are frequently used to detect and quantify TNFRSF10B protein expression in cell and tissue lysates. The search results demonstrate successful detection in multiple cancer cell lines, including HepG2 (hepatocellular carcinoma) and HCT-116 (colorectal carcinoma), where the protein typically appears as bands at approximately 44-52 kDa . Different antibody clones may require specific buffer systems for optimal results, such as Immunoblot Buffer Group 2 for MAB6313 or Buffer Group 8 for AF631 .

Neutralization Studies: Neutralizing antibodies against TNFRSF10B block the interaction between TRAIL and its receptor, inhibiting TRAIL-induced apoptosis. These antibodies are valuable for mechanistic studies investigating the contribution of TRAIL-TNFRSF10B signaling in various biological processes. The typical concentration range for effective neutralization is approximately 7.00-70.0 ng/mL .

Functional Assays: Antibodies targeting TNFRSF10B can be used to study apoptosis sensitivity in various cell types. Research has demonstrated that "an antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells" , suggesting similar approaches may be applicable for TNFRSF10B-targeted strategies.

Immunohistochemistry/Immunocytochemistry: Though not explicitly described in the search results, TNFRSF10B antibodies are commonly employed to examine receptor expression and localization in tissue sections or cultured cells, providing spatial information about receptor distribution.

Therapeutic Development: The search results highlight that "TNFRSF agonistic antibodies have been evaluated extensively in preclinical models," with their robust antitumor immune responses encouraging continued clinical investigations . These approaches aim to harness the apoptosis-inducing potential of TNFRSF10B for cancer treatment.

What are the optimal conditions for detecting TNFRSF10B in Western Blot applications?

Successful detection of TNFRSF10B via Western blot requires careful optimization of multiple experimental parameters to ensure specific and reproducible results. Based on the search results, the following conditions have been validated for effective TNFRSF10B detection:

Sample Preparation: TNFRSF10B is optimally detected under reducing conditions, as demonstrated in the protocols for both monoclonal (MAB6313, MAB631) and polyclonal (AF631) antibodies . The choice of lysis buffer significantly impacts detection efficiency, with specific buffer systems recommended for different antibody clones—Immunoblot Buffer Group 2 for MAB6313 and Buffer Group 8 for AF631 .

Antibody Selection and Concentration: The search results consistently report effective primary antibody concentrations of approximately 1 μg/mL for both monoclonal and polyclonal anti-TNFRSF10B antibodies . Secondary antibody selection should match the host species of the primary antibody (e.g., HRP-conjugated Anti-Mouse IgG for mouse monoclonal antibodies like MAB6313, or HRP-conjugated Anti-Goat IgG for goat polyclonal antibodies like AF631) .

Membrane Selection: PVDF membranes have been successfully used for TNFRSF10B detection in all reported protocols . This membrane type offers appropriate protein binding capacity and background characteristics for detecting membrane proteins like TNFRSF10B.

Expected Banding Pattern: Researchers should anticipate TNFRSF10B to appear as bands at approximately 44-52 kDa, with potential variability attributable to different glycosylation states or other post-translational modifications . Multiple bands within this range may represent different modified forms rather than non-specific binding.

Positive Control Selection: HepG2 (hepatocellular carcinoma) and HCT-116 (colorectal carcinoma) cell lines serve as reliable positive controls for TNFRSF10B detection, as they consistently express detectable levels of the protein .

Optimization Considerations: If signal strength is insufficient, researchers might consider extending incubation times or slightly increasing antibody concentration. Conversely, high background might necessitate more stringent washing protocols or reduced antibody concentration.

How do neutralizing antibodies against TNFRSF10B affect TRAIL-induced apoptosis?

Neutralizing antibodies targeting TNFRSF10B interfere with TRAIL-induced apoptosis through specific molecular mechanisms that have important implications for both basic research and therapeutic development. Understanding these effects requires consideration of several key aspects:

Molecular Mechanism: Neutralizing antibodies bind to the extracellular domain of TNFRSF10B, typically at or near the TRAIL-binding interface. This physical obstruction prevents TRAIL from engaging with its receptor, thereby inhibiting the conformational changes and receptor clustering necessary for death-inducing signaling complex (DISC) formation. The search results specifically note that "The human TRAIL R2/Fc chimera neutralizes the ability of TRAIL to induce apoptosis" , demonstrating this principle.

Effective Concentration Range: According to the search results, the concentration range for effective neutralization is typically 7.00-70.0 ng/mL . This relatively low concentration highlights the high affinity and specificity of well-designed neutralizing antibodies.

Experimental Applications: Neutralizing antibodies serve as valuable tools for dissecting the specific contribution of TNFRSF10B in TRAIL-induced apoptosis, particularly in systems where multiple TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) are expressed. By selectively blocking one receptor subtype, researchers can determine the relative importance of each receptor in different cellular contexts.

Cellular Context Considerations: The effectiveness of neutralizing antibodies may vary depending on several factors, including receptor expression levels, the presence of decoy receptors (TRAIL-R3/DcR1 and TRAIL-R4/DcR2), and the activation state of downstream signaling pathways. In cells with high expression of decoy receptors, the effect of TNFRSF10B neutralization might be less pronounced due to already compromised TRAIL signaling.

Combination Studies: Neutralizing antibodies can reveal how TRAIL signaling interacts with other apoptotic stimuli. Research indicates that antibodies against death receptors can modulate the effects of chemotherapeutic agents, as seen in studies where "an antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells" . Similar principles may apply to TNFRSF10B targeting.

What are the challenges in distinguishing TNFRSF10B from other TRAIL receptors?

Distinguishing TNFRSF10B (TRAIL-R2/DR5) from other members of the TRAIL receptor family presents significant technical and experimental challenges that researchers must address for accurate characterization and functional studies:

Sequence Homology: TNFRSF10B shares 55% amino acid sequence identity with TNFRSF10A (TRAIL-R1/DR4) , creating substantial potential for antibody cross-reactivity. This homology is particularly pronounced in the extracellular domains that bind TRAIL, making it challenging to develop antibodies that discriminate perfectly between these receptors.

Antibody Validation Requirements: Confirming antibody specificity necessitates rigorous testing against all TRAIL receptor family members. Ideal validation would include cells engineered to express only one receptor type through genetic manipulation. The search results do not explicitly address validation strategies, highlighting a potential gap in standard protocols.

Functional Redundancy: Both TNFRSF10A and TNFRSF10B signal for apoptosis upon TRAIL binding, while decoy receptors (TRAIL-R3/DcR1 and TRAIL-R4/DcR2) compete for the same ligand without inducing apoptosis. This functional overlap complicates the interpretation of studies using neutralizing or agonistic antibodies, as the observed effects might represent the combined influence of multiple receptors.

Detection Method Challenges: In Western blotting applications, TRAIL receptors have similar molecular weights (TNFRSF10B appears at approximately 44-52 kDa ), necessitating high-resolution gels for adequate separation. Flow cytometry and immunohistochemistry face similar challenges due to potential cross-reactivity of antibodies against these structurally similar proteins.

Strategies for Improved Specificity: Several approaches can enhance receptor discrimination, including the use of receptor-selective agonists or antibodies that target unique epitopes, genetic tools like CRISPR/Cas9 or siRNA to knock down individual receptors as controls, and combined approaches that integrate data from multiple techniques (e.g., qPCR for mRNA plus protein detection).

Reporting Standards: Given these challenges, comprehensive reporting of antibody validation methods, clone information, and potential limitations is essential for research reproducibility. Studies should clearly state which receptor isoform was targeted and how specificity was confirmed.

How do post-translational modifications affect TNFRSF10B detection?

Post-translational modifications (PTMs) of TNFRSF10B significantly impact antibody detection, protein function, and experimental interpretation. Understanding these effects is crucial for accurate characterization and functional analysis:

Glycosylation Effects: TNFRSF10B contains N-linked glycosylation sites that contribute to its molecular weight heterogeneity. The Western blot results demonstrate TNFRSF10B appearing at approximately 44-52 kDa , with this variation likely reflecting different glycosylation states. Epitopes located near glycosylation sites may be masked, affecting antibody binding efficiency. Deglycosylation treatments (e.g., PNGase F) can help confirm that higher molecular weight bands represent glycosylated forms rather than non-specific binding.

Phosphorylation Considerations: The cytoplasmic domain of TNFRSF10B contains potential phosphorylation sites that may regulate its signaling activity. When studying phosphorylation states, phosphatase inhibitors should be included in lysis buffers to preserve these modifications. Treatment with phosphatase before Western blot can help determine if band shifts are due to phosphorylation events.

Ubiquitination and Degradation: TNFRSF10B undergoes ubiquitin-mediated regulation affecting its stability and trafficking. Detecting ubiquitinated forms may require proteasome inhibitors (e.g., MG132) and modified extraction protocols. Antibodies targeting different domains may show varying ability to detect ubiquitinated TNFRSF10B.

Experimental Approaches: To address PTM challenges, researchers should consider using multiple antibodies targeting different epitopes, include PTM-modifying treatments in parallel samples, and carefully document running conditions as PTMs can alter migration patterns in electrophoresis. The search results indicate that different buffer systems may be optimal for different antibody clones (e.g., Immunoblot Buffer Group 2 for MAB6313 or Buffer Group 8 for AF631) , which may relate to preservation of relevant PTMs.

Functional Implications: PTMs may affect TRAIL binding affinity and downstream signaling. When studying TNFRSF10B function, considering the PTM status is crucial for interpreting results. Cellular stress, activation states, and drug treatments can alter the PTM profile of TNFRSF10B, potentially confounding experimental outcomes if not properly controlled.

What are the implications of TNFRSF10B in cancer immunotherapy?

TNFRSF10B has emerged as a significant target for cancer immunotherapy, with several important implications for therapeutic development:

Direct Apoptosis Induction: Agonistic antibodies against TNFRSF10B can trigger cancer cell death directly through the extrinsic apoptotic pathway. These antibodies mimic the action of TRAIL but may offer improved pharmacokinetics and reduced off-target effects. The search results indicate that similar approaches with related receptors have shown promising results: "An antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells" .

Advanced Targeting Approaches: "Cross-linking through tumor antigen binding with bispecific antibodies" represents a frontier approach in TNFRSF10B-targeted therapy. This strategy involves designing antibodies where one arm targets TNFRSF10B while the other targets a tumor-specific antigen, potentially localizing TNFRSF10B activation to the tumor microenvironment for improved safety and efficacy.

Combination Therapy Opportunities: TNFRSF10B agonists may sensitize cancer cells to conventional treatments like chemotherapy or radiation. The search results suggest this approach with "antibody against DR4 (TRAIL-R1) in combination with doxorubicin" , and similar principles likely apply to TNFRSF10B targeting. Additionally, TNFRSF10B activation could complement immune checkpoint blockade by directly eliminating cancer cells.

Challenges in Clinical Translation: "Balancing the toxicities and efficacy of TNFRSF agonistic antibodies remains a major challenge in the clinical development" . This highlights the importance of developing strategies that enhance tumor-specific activity while minimizing systemic effects on normal tissues expressing TNFRSF10B. The research literature suggests that "leveraging the interactions between antibodies and the inhibitory Fc receptor FcγRIIB to optimize co-stimulation agonistic activities dependent on FcγRIIB cross-linking selectively in tumor microenvironment represents the current frontier" .

How can TNFRSF10B antibodies be validated for specificity?

Thorough validation of TNFRSF10B antibodies is essential for ensuring experimental reliability and reproducibility. A comprehensive validation strategy should include multiple complementary approaches:

Genetic Approaches: The most definitive validation utilizes genetic manipulation of TNFRSF10B expression. Testing antibodies on TNFRSF10B knockout cell lines (CRISPR/Cas9-generated) or after siRNA-mediated knockdown should result in abolished or significantly reduced signal. Conversely, comparing antibody signal in wild-type cells versus those overexpressing TNFRSF10B confirms the antibody detects the target when present at higher levels.

Biochemical Validation: Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, provide evidence of epitope specificity—the specific signal should be blocked. Immunoprecipitation followed by mass spectrometry analysis can confirm that TNFRSF10B is the predominant protein recognized by the antibody.

Cross-Reactivity Assessment: Given that TNFRSF10B shares 55% sequence homology with TNFRSF10A (DR4) , evaluating potential cross-reactivity is crucial. This assessment should involve testing against recombinant proteins or cells selectively expressing each TRAIL receptor type. Species specificity should also be documented if working with non-human models.

Application-Specific Validation: For Western blot applications, researchers should confirm the expected molecular weight (approximately 44-52 kDa as shown in the search results ) and document reducing versus non-reducing conditions. For functional validation of neutralizing or agonistic antibodies, dose-dependent effects on TRAIL-induced apoptosis should be confirmed, and specificity of effect tested using cells lacking TNFRSF10B.

Multi-technique Confirmation: Verification of findings using different detection methods (e.g., Western blot, immunofluorescence, flow cytometry) increases confidence in antibody specificity. Additionally, using multiple antibodies targeting different epitopes of TNFRSF10B with concordant results strongly supports specificity of detection.

Documentation Standards: Comprehensive reporting of validation methodology in publications, including catalog numbers, lot numbers, and dilutions used, is essential for research reproducibility. Any limitations observed during validation should be transparently documented to guide other researchers.

What are the best experimental controls when working with TNFRSF10B antibodies?

Robust experimental controls are essential for ensuring the validity and interpretability of research involving TNFRSF10B antibodies. A comprehensive control strategy should include:

Positive Controls: Cell lines with confirmed TNFRSF10B expression provide essential references for antibody performance. Based on the search results, HepG2 (hepatocellular carcinoma) and HCT-116 (colorectal carcinoma) cell lines serve as suitable positive controls for Western blot applications . Recombinant TNFRSF10B protein at known concentrations can help establish detection limits and antibody sensitivity. Overexpression systems using cells transfected with TNFRSF10B expression constructs provide strong positive controls, particularly useful for antibody validation.

Negative Controls: TNFRSF10B knockout or knockdown cells (using CRISPR/Cas9 or siRNA technology) represent ideal negative controls to confirm signal specificity. Secondary antibody-only controls are essential for determining background signal in immunodetection methods. Isotype control antibodies are particularly important for flow cytometry and functional studies to control for non-specific Fc receptor interactions.

Specificity Controls: Peptide competition/blocking experiments, where pre-incubation of the antibody with the immunizing peptide should abolish specific staining, provide compelling evidence of epitope specificity. Cross-reactivity assessment against related proteins (especially TNFRSF10A/DR4, given the 55% sequence homology ) confirms target selectivity. Using different antibodies targeting distinct epitopes of TNFRSF10B to confirm findings provides additional validation.

Functional Controls: For neutralizing antibodies, parallel experiments with known TRAIL pathway inhibitors (e.g., caspase inhibitors) help confirm the specificity of observed effects. For agonistic antibodies, comparison with recombinant TRAIL establishes a benchmark for activity. Dose-response analysis using a range of antibody concentrations helps establish optimal working conditions and demonstrate specific effects.

Technical Controls: Loading controls for Western blot (e.g., β-actin, GAPDH) ensure equal protein loading across samples. Standardized positive samples across experiments monitor inter-assay variability. Lot-to-lot comparison when receiving new antibody batches helps maintain consistent experimental conditions.

How does the binding epitope affect the functionality of TNFRSF10B antibodies?

The binding epitope of TNFRSF10B antibodies significantly influences their functional properties, with important implications for research applications and therapeutic development:

Epitope Location Effects: Antibodies binding to different regions of TNFRSF10B exhibit distinct functional characteristics. Those targeting the TRAIL-binding region (extracellular domain) can function as neutralizing antibodies by preventing ligand-receptor interaction. The search results reference antibodies targeting regions like "Ala54-Glu182" or "Ile56-Pro128" , which likely encompass the ligand-binding domain. In contrast, antibodies binding to other epitopes may allow TRAIL binding while affecting downstream signaling events or receptor clustering.

Agonistic Activity Determinants: The search results explicitly state that "the binding epitope of the TNFRSF target is one of the key factors determining the intrinsic agonistic activities of the antibody" . Certain epitopes allow antibodies to mimic TRAIL binding, inducing receptor clustering and signaling that promotes apoptosis. Antibodies binding to these epitopes can trigger conformational changes that facilitate death-inducing signaling complex (DISC) formation.

Spatial Arrangement Considerations: The orientation of bound antibodies affects the geometry of receptor clustering, with optimal signaling requiring specific spatial arrangements of the intracellular death domains. Different epitopes may facilitate or hinder this optimal arrangement, directly impacting signaling efficiency.

Therapeutic Implications: Understanding epitope-function relationships informs rational antibody design for therapeutic applications. "Insights into the co-stimulation signaling biology, antibody structural roles and their functionality in immuno-oncology are guiding new advancement" in this field. Advanced approaches like "cross-linking antibody (xLinkAb) model and its application in developing TNFRSF agonistic antibodies" leverage these principles to enhance efficacy and safety profiles.