Recombinant Human Tumor necrosis factor receptor superfamily member 17 protein (TNFRSF17), partial (Active)

What is TNFRSF17 and what are its alternate names in scientific literature?

TNFRSF17, also known as B-cell maturation antigen (BCMA), BCM, CD269, or TNFRSF13A, is a cell surface receptor belonging to the tumor necrosis factor receptor superfamily. It is encoded by the TNFRSF17 gene in humans and primarily expressed on mature B lymphocytes . The protein functions as a receptor that recognizes B-cell activating factor (BAFF) and plays significant roles in B-cell development, autoimmune responses, and various pathological conditions . In scientific research, it's important to search literature using all variant names to ensure comprehensive coverage of relevant publications.

How is the recombinant partial TNFRSF17 protein structurally different from the full-length native protein?

The recombinant partial TNFRSF17 protein typically contains the extracellular domain (such as amino acids 1-54) fused with an hFc-tag at the C-terminal . This differs from the full-length native protein which includes the transmembrane and intracellular domains necessary for signal transduction. The partial recombinant protein maintains the binding domain (BCMA TALL-1 binding domain) required for interaction with its ligand TNFSF13B , but lacks the ability to independently initiate downstream signaling pathways like NF-kappaB and MAPK8/JNK. The molecular weight of the partial recombinant protein (approximately 34.8 kDa) differs from the native protein due to the truncation and addition of tags designed to facilitate purification and detection in experimental settings .

What are the key functional domains of TNFRSF17 and how do they influence experimental design?

The key functional domain of TNFRSF17 is the BCMA TALL-1 binding domain located at the N-terminus, which is required for binding to TNFSF13B (BAFF) . This conserved domain is crucial for the protein's biological activity. When designing experiments involving TNFRSF17, researchers must ensure that this domain remains intact and properly folded in recombinant constructs.

For experimental design, considerations should include:

• Verifying binding capability through functional assays (ELISA, LSPR) with known ligands

• Ensuring proper protein folding by quality control methods

• Using appropriate buffer conditions to maintain structural integrity

• Determining whether the experimental question requires the full signaling capability (needing full-length protein) or just binding interactions (where partial protein may suffice)

• Including appropriate controls to distinguish effects of the protein from those of any fusion tags

How can recombinant TNFRSF17 be used as a standard in ELISA-based detection methods?

Recombinant TNFRSF17 serves as an excellent standard in ELISA-based detection methods due to its high purity and well-characterized binding properties. To implement TNFRSF17 as an ELISA standard:

• Coat ELISA plates with purified recombinant TNFRSF17 at varying concentrations (typically 0.1-10 μg/ml) to establish a standard curve

• Block non-specific binding sites with appropriate blocking buffer

• Add the ligand (such as biotinylated TNFSF13B) followed by detection reagents

• Analyze binding curves to determine EC50 values (typically in the range of 0.31-0.57 ng/ml for high-quality preparations)

For quantitative detection of TNFRSF17 in patient samples, the recombinant protein provides a reliable reference point for calibration. When developing such assays, researchers should verify batch consistency through quality control measures including SDS-PAGE analysis for purity (>90% recommended) and endotoxin testing (<1.0 EU/μg using LAL method) to ensure reliable and reproducible results .

What are the optimal experimental conditions for studying TNFRSF17 interactions with its ligands?

The optimal experimental conditions for studying TNFRSF17 interactions with its ligands (particularly TNFSF13B/BAFF) include:

Buffer composition:

• Physiological pH (7.2-7.4)

• PBS or TBS with 0.05-0.1% non-ionic detergent for reduced non-specific binding

• 1-5% BSA or similar blocking protein

• Calcium (1-2 mM) to maintain protein stability

Analytical techniques:

• Surface Plasmon Resonance (SPR) or LSPR for real-time binding kinetics

• ELISA for endpoint measurements

• Flow cytometry for cell-surface expression studies

• Co-immunoprecipitation for complex formation analysis

Important controls:

• Negative control using non-relevant proteins of similar size

• Competitive binding assays using known ligands

• Validation with multiple techniques to confirm specificity

Researchers should maintain sample integrity by avoiding freeze-thaw cycles and using freshly prepared reagents when possible. For kinetic studies, temperature control (typically 25°C or 37°C) is essential for reproducible results .

How can researchers effectively use recombinant TNFRSF17 in cell-based assays to study B-cell function?

To effectively use recombinant TNFRSF17 in cell-based assays studying B-cell function:

• Cell selection: Primary B cells, plasma cells, or B-cell lines (such as those derived from multiple myeloma) that express the complementary ligands

• Assay formats:

  • Stimulation assays: Use plate-bound or soluble recombinant TNFRSF17 to stimulate cells expressing TNFSF13B

  • Inhibition assays: Use recombinant TNFRSF17 to competitively inhibit TNFSF13B-TNFRSF17 interactions

  • Cell viability/proliferation assays: Measure effects on B-cell survival using MTT, XTT, or flow cytometry

• Functional readouts:

  • NF-κB activation (using reporter cell lines or Western blotting)

  • MAPK8/JNK pathway activation

  • Changes in gene expression profiles

  • B-cell survival and proliferation metrics

  • Antibody production measurement

• Optimization strategies:

  • Titrate protein concentrations (typically 1-100 ng/ml)

  • Determine optimal time points for measuring different responses

  • Include pathway inhibitors as controls to confirm specificity

When designing these experiments, researchers should consider the partial nature of recombinant TNFRSF17 proteins which may affect certain signaling outcomes compared to the native receptor .

How is TNFRSF17 expression altered in multiple myeloma and what implications does this have for therapeutic approaches?

TNFRSF17 expression is significantly upregulated in multiple myeloma (MM) compared to normal plasma cells. Specifically:

• Expression patterns:

  • Elevated surface expression on malignant plasma cells

  • Increased levels of soluble BCMA (sBCMA, the cleaved form of TNFRSF17) in serum of MM patients

  • Expression correlates with disease progression and relapse

• Therapeutic implications:

  • TNFRSF17 serves as a target for various immunotherapies including:

    • CAR T-cell therapies

    • Antibody-drug conjugates (e.g., Belantamab mafodotin)

    • T-cell engagers (TCEs)

• Resistance mechanisms:

  • Biallelic deletion of TNFRSF17 (TNFSF17) has been identified as a rare but important mechanism of antigen escape and resistance to BCMA-targeted therapies

  • Extracellular domain missense mutations (e.g., R27P) can mediate resistance to T-cell engagers

  • Preexisting subclonal TNFRSF17 deletion (occurring in approximately 5% of patients prior to CAR T-cell therapy) may predict treatment resistance

These findings underscore the need for monitoring TNFRSF17 structural changes and expression levels before and during treatment. Combination approaches targeting multiple epitopes or alternative B-cell markers may help overcome resistance mechanisms in clinical settings .

What is the role of TNFRSF17 in lupus nephritis and how might it be targeted therapeutically?

TNFRSF17 plays a significant role in lupus nephritis (LN) pathogenesis with strong therapeutic implications:

• Expression patterns:

  • Significantly overexpressed in both peripheral blood mononuclear cells (PBMCs) and kidney tissue of LN patients

  • Distributed in glomeruli, tubules, and interstitium of LN patient kidneys

  • Expression particularly elevated in class III, IV, and V LN

• Clinical correlations:

  • Positively correlated with 24-hour urine protein quantification (marker of disease severity)

  • Negatively correlated with complement C3 and C4 levels

  • Associated with extrafollicular B cell responses marked by expansion of atypical B cells and antibody-secreting cells

• Potential therapeutic approaches:

  • TNFRSF17-targeting drugs like IBI379 (a construct with anti-TNFRSF17 chains and anti-CD3-ScFv-Fc fusion)

  • These agents can effectively induce apoptosis in patient plasma cells with minimal effects on B cells

  • May address the extrafollicular B cell response characteristic of childhood idiopathic nephrotic syndrome

Mechanistically, TNFRSF17 mediates plasma cell survival through the classical NF-κB pathway, suggesting that targeting this receptor could disrupt pathogenic antibody production. The specific correlation with disease markers and the demonstrated efficacy of experimental TNFRSF17-targeting agents makes this receptor a promising therapeutic target for lupus nephritis .

How can researchers detect and quantify TNFRSF17 deletion and mutations in patient samples?

Researchers can effectively detect and quantify TNFRSF17 deletion and mutations in patient samples using several complementary approaches:

• Genomic analysis:

  • Whole-genome sequencing (WGS) with 100x coverage on paired normal and tumor cells

  • Targeted next-generation sequencing of the TNFRSF17 locus

  • Copy number variation (CNV) analysis to detect deletions at chromosome 16

• Single-cell approaches:

  • Single-cell mRNA analysis to detect expression at cellular resolution

  • Single-cell CNV analysis to identify subclonal populations with TNFRSF17 deletion

• Protein detection:

  • Flow cytometry for surface expression quantification

  • Immunohistochemistry for tissue localization

  • ELISA for soluble/cleaved forms (sBCMA)

• Mutation analysis:

  • Targeted sequencing of the extracellular domain to identify mutations (e.g., R27P)

  • Functional validation of identified mutations using binding assays

For clinical relevance, researchers should monitor TNFRSF17 status before treatment initiation and at relapse, particularly in patients receiving BCMA-targeted therapies. Sensitivity is crucial—techniques should detect subclonal deletions with variant allelic fractions as low as 3.5%, as these can impact treatment response . When analyzing results, researchers should consider the size of deletions (approximately 0.8 Mb pairs encompassing the TNFRSF17 gene locus has been reported) and distinguish between monoallelic and biallelic losses .

How can researchers develop multi-epitope targeting approaches to overcome TNFRSF17 antigen escape in immunotherapies?

Developing multi-epitope targeting approaches to overcome TNFRSF17 antigen escape requires systematic strategies:

• Epitope mapping and selection:

  • Identify conserved epitopes across the TNFRSF17 protein using crystallographic data

  • Target multiple non-overlapping epitopes simultaneously

  • Include epitopes from both N-terminal and C-terminal regions

  • Focus on epitopes critical for ligand binding or signaling

• Combination therapy design:

  • Develop cocktails of monoclonal antibodies targeting different TNFRSF17 epitopes

  • Create bispecific or multispecific antibodies that engage TNFRSF17 and additional B-cell markers

  • Combine TNFRSF17-targeted therapy with agents targeting alternative B-cell pathways

• CAR T-cell engineering approaches:

  • Design CAR T-cells with multiple single-chain variable fragments recognizing different TNFRSF17 epitopes

  • Develop dual-targeting CAR T-cells recognizing both TNFRSF17 and secondary targets (e.g., CD38, SLAMF7)

  • Incorporate logic-gated CAR designs requiring recognition of multiple antigens

• Monitoring and adaptation strategies:

  • Implement sequential liquid biopsies to detect emerging TNFRSF17 mutations or deletions

  • Develop real-time epitope escape monitoring protocols

  • Design adaptive treatment protocols that modify targeting strategy based on molecular monitoring

This comprehensive approach acknowledges that biallelic deletion of TNFRSF17 and extracellular domain mutations represent important mechanisms of resistance to BCMA-targeted therapies. By simultaneously targeting multiple epitopes or combining TNFRSF17 targeting with other approaches, researchers can potentially prevent or overcome antigen escape phenomena .

What experimental approaches can determine the functional consequences of TNFRSF17 missense mutations on binding to therapeutic antibodies?

To determine the functional consequences of TNFRSF17 missense mutations on binding to therapeutic antibodies, researchers should employ a systematic multi-technique approach:

• Structural analysis:

  • Computational modeling of mutations using protein structure prediction tools

  • X-ray crystallography or cryo-EM of mutant-antibody complexes

  • Molecular dynamics simulations to predict binding energy changes

• Binding kinetics assessment:

  • Surface plasmon resonance (SPR) comparing wild-type and mutant TNFRSF17 binding to therapeutic antibodies

  • Bio-layer interferometry to determine kon and koff rates

  • Isothermal titration calorimetry for thermodynamic binding parameters

• Cell-based functional assays:

  • Generate cell lines expressing wild-type or mutant TNFRSF17 (e.g., R27P)

  • Flow cytometry to quantify antibody binding to cell surface

  • Antibody-dependent cellular cytotoxicity (ADCC) assays

  • Complement-dependent cytotoxicity (CDC) assays

  • Cell killing assays with antibody-drug conjugates or T-cell engagers

• In vitro mutation screening:

  • Display libraries (phage, yeast) of TNFRSF17 variants

  • High-throughput screening for escape mutations

  • Deep mutational scanning to comprehensively map mutation effects

• Validation in patient-derived samples:

  • Correlate identified mutations with clinical response data

  • Ex vivo testing of therapeutic antibodies on patient samples with known mutations

This comprehensive approach allows researchers to identify which mutations significantly impact therapeutic binding, understand the structural basis for resistance, and potentially design next-generation therapeutics that maintain efficacy against mutant forms of TNFRSF17 .

How can recombinant TNFRSF17 be used to study the extrafollicular B cell response in autoimmune conditions?

Recombinant TNFRSF17 offers valuable tools for studying extrafollicular B cell responses in autoimmune conditions through several methodological approaches:

• Ex vivo analysis of patient samples:

  • Use labeled recombinant TNFRSF17 to identify cells expressing complementary ligands

  • Develop flow cytometry panels incorporating TNFRSF17 binding to characterize extrafollicular B cell subsets

  • Perform competitive binding assays with patient serum to detect autoantibodies targeting TNFRSF17-related pathways

• In vitro functional studies:

  • Culture sorted B cell populations with recombinant TNFRSF17 to assess effects on differentiation

  • Analyze changes in gene expression profiles using RNA-seq after TNFRSF17 stimulation

  • Compare responses between healthy donor and autoimmune patient B cells

• Signaling pathway interrogation:

  • Use recombinant TNFRSF17 to activate or inhibit signaling in various B cell subsets

  • Assess NF-κB and MAPK8/JNK pathway activation in extrafollicular vs. follicular B cells

  • Study the role of TNFRSF17 in plasma cell survival and antibody production

• Animal model applications:

  • Administer recombinant TNFRSF17 to rodent models of autoimmunity (e.g., NZB/W mice)

  • Track effects on extrafollicular B cell expansion and autoantibody production

  • Test TNFRSF17-targeting strategies in these models

This approach is particularly relevant given recent findings that extrafollicular B cell responses, marked by expansion of atypical B cells and antibody-secreting cells, represent the major immune perturbation in conditions like childhood idiopathic nephrotic syndrome and lupus nephritis. TNFRSF17 expression has been found to correlate positively with disease markers such as urinary protein levels, suggesting its central role in these pathogenic processes .

What are the critical quality control parameters for validating recombinant TNFRSF17 activity in research applications?

Critical quality control parameters for validating recombinant TNFRSF17 activity include:

• Purity assessment:

  • SDS-PAGE analysis to confirm >90% purity

  • Mass spectrometry to verify protein identity and molecular weight (~34.8 kDa for partial constructs with tags)

  • Host cell protein (HCP) quantification with target levels below 100 ppm

• Endotoxin testing:

  • LAL method with acceptable limits <1.0 EU/μg

  • Ensure samples are endotoxin-free to prevent experimental artifacts

• Functional validation:

  • Binding activity assessment through ELISA with established ligands (e.g., TNFSF13B)

  • Determination of EC50 values (typical range: 0.31-0.57 ng/ml for high-quality preparations)

  • LSPR assay to confirm binding kinetics

• Structural integrity:

  • Circular dichroism to verify proper protein folding

  • Size exclusion chromatography to detect aggregation

  • Thermal shift assays to assess stability

• Batch consistency:

  • Lot-to-lot comparison of activity using standardized assays

  • Reference standard inclusion in validation experiments

  • Certificate of analysis documenting critical parameters

These quality control measures ensure that experimental outcomes reflect true biological activity rather than artifacts from impurities, misfolding, or degradation. For researchers conducting critical experiments, validating multiple parameters rather than relying on a single quality metric provides greater confidence in results and improves reproducibility across laboratories .

How can researchers distinguish between the effects of full-length and partial TNFRSF17 in experimental systems?

Distinguishing between the effects of full-length and partial TNFRSF17 in experimental systems requires strategic experimental design:

• Complementary expression systems:

  • Generate matched cell lines expressing either full-length or partial TNFRSF17

  • Ensure equivalent expression levels through quantitative flow cytometry

  • Use inducible expression systems for temporal control

• Domain-specific functional assays:

  • Binding assays: Both full-length and partial proteins should demonstrate comparable ligand binding

  • Signaling assays: Only full-length protein will initiate downstream signaling cascades

  • Monitor NF-κB and MAPK8/JNK activation using reporter systems or phosphorylation studies

• Competitive experimental designs:

  • Use partial protein as a competitive inhibitor of full-length protein

  • Titrate partial protein to determine binding site saturation effects

  • Analyze dose-response relationships for both variants

• Molecular controls:

  • Generate domain deletion mutants to pinpoint functional regions

  • Create chimeric constructs swapping domains between TNFRSF17 and related receptors

  • Use site-directed mutagenesis to disrupt specific functions while preserving others

• Readout selection:

  • Select proximal readouts (e.g., receptor clustering) and distal readouts (e.g., gene expression)

  • Time-course experiments to distinguish immediate vs. delayed effects

  • Single-cell analyses to capture heterogeneous responses

By implementing these strategies, researchers can clearly delineate which experimental observations are attributable to the binding/competitive functions of the partial protein versus the complete signaling capabilities of the full-length receptor .

What are the best practices for handling and storing recombinant TNFRSF17 to maintain optimal activity?

Best practices for handling and storing recombinant TNFRSF17 to maintain optimal activity include:

• Storage conditions:

  • Store lyophilized protein at -20°C or -80°C

  • After reconstitution, aliquot and store at -80°C to minimize freeze-thaw cycles

  • Avoid storage in frost-free freezers due to temperature fluctuations

• Reconstitution protocol:

  • Use sterile, molecular biology-grade water or buffer

  • For higher concentration needs, reconstitute in minimal volume and further dilute in working buffer

  • Allow protein to fully dissolve (typically 10-20 minutes at room temperature) with gentle swirling rather than vortexing

• Working solution preparation:

  • Optimal buffer composition: PBS or TBS with 0.1% carrier protein (BSA or HSA)

  • pH range: 7.2-7.4

  • Optional additives: 0.05% sodium azide for extended storage, protease inhibitors for sensitive applications

  • Filter-sterilize working solutions (0.22 μm filter)

• Stability considerations:

  • Minimize freeze-thaw cycles (ideally ≤3)

  • Maintain protein at 2-8°C during experiment preparation (not room temperature)

  • Discard diluted working solutions after 24 hours

  • Monitor solution clarity; cloudiness indicates potential aggregation

• Quality verification before critical experiments:

  • Perform binding activity check using simple ELISA

  • Verify protein concentration using standardized methods

  • Consider including positive control from previously validated lot

Following these practices ensures that experimental outcomes reflect true biological activity rather than artifacts from protein degradation or denaturation. For critical applications, researchers should document handling procedures and include appropriate controls to verify protein functionality at the time of experiment .

How might single-cell technologies enhance our understanding of TNFRSF17 expression heterogeneity in disease states?

Single-cell technologies offer powerful approaches to understand TNFRSF17 expression heterogeneity in disease states:

• Single-cell RNA sequencing applications:

  • Identify previously unrecognized cell populations with differential TNFRSF17 expression

  • Map TNFRSF17 expression across B-cell differentiation trajectories

  • Correlate TNFRSF17 expression with broader transcriptional programs

  • Detect rare resistant cell populations before they expand under therapeutic pressure

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) to simultaneously measure TNFRSF17 and dozens of other proteins

  • Imaging mass cytometry for spatial context in tissue samples

  • Single-cell Western blotting for protein isoform discrimination

• Genomic heterogeneity assessment:

  • Single-cell DNA sequencing to detect copy number variations in TNFRSF17

  • G&T-seq (genome and transcriptome sequencing) to directly link genetic alterations to expression changes

  • CRISPR screening at single-cell resolution to identify regulators of TNFRSF17 expression

• Integrated multi-omics approaches:

  • CITE-seq to simultaneously measure surface TNFRSF17 protein and mRNA expression

  • Single-cell ATAC-seq to determine chromatin accessibility at the TNFRSF17 locus

  • Spatial transcriptomics to map TNFRSF17 expression in tissue context

Recent findings using these technologies have already revealed important insights, such as identifying the extrafollicular B cell response as a major immune perturbation in childhood idiopathic nephrotic syndrome, characterized by expansion of atypical B cells and antibody-secreting cells with distinctive TNFRSF17 expression patterns . Similarly, single-cell approaches have detected rare subclonal TNFRSF17 deletions in approximately 5% of multiple myeloma patients prior to CAR T-cell therapy, with significant implications for treatment response .

What are the emerging approaches for developing next-generation TNFRSF17-targeted therapeutics?

Emerging approaches for developing next-generation TNFRSF17-targeted therapeutics include:

• Novel antibody engineering:

  • Bispecific antibodies targeting TNFRSF17 and CD3 (e.g., IBI379)

  • Antibodies targeting conserved epitopes resistant to common mutations

  • pH-dependent binding antibodies for improved internalization

  • Antibody mixtures targeting multiple TNFRSF17 epitopes simultaneously

• Advanced cellular therapies:

  • CRISPR-engineered CAR T-cells with enhanced persistence

  • Dual-targeting CAR T-cells recognizing TNFRSF17 and secondary targets

  • Logic-gated CAR designs requiring recognition of multiple antigens

  • Natural killer (NK) cell-based therapies targeting TNFRSF17

• Innovative conjugate technologies:

  • Site-specific conjugation methods for improved ADC homogeneity

  • Novel payload classes beyond traditional cytotoxins

  • Conditionally activated ADCs that release payload only in tumor microenvironment

  • Increased drug-antibody ratios with maintained stability

• Alternative modalities:

  • Proteolysis targeting chimeras (PROTACs) directing TNFRSF17 for degradation

  • mRNA-encoded TNFRSF17-targeting therapeutics

  • Small molecule degraders of TNFRSF17

  • Engineered TNFSF13B variants with enhanced binding

• Predictive biomarker integration:

  • Real-time monitoring of TNFRSF17 expression and mutations

  • Adaptive treatment protocols based on molecular monitoring

  • Companion diagnostics to identify patients most likely to benefit

These approaches address key limitations of current TNFRSF17-targeted therapies, including antigen escape through TNFRSF17 deletion or mutation, limited durability of response, and on-target off-tumor toxicity. Particularly promising are combinations that can effectively induce apoptosis in malignant or pathogenic plasma cells while minimizing effects on normal B cells, as demonstrated with agents like IBI379 in lupus nephritis models .

How can systems biology approaches integrate TNFRSF17 signaling into broader B cell regulatory networks?

Systems biology approaches offer powerful frameworks for integrating TNFRSF17 signaling into broader B cell regulatory networks:

• Network modeling strategies:

  • Construct protein-protein interaction networks centered on TNFRSF17

  • Develop dynamic mathematical models of TNFRSF17 signaling pathways (NF-κB and MAPK8/JNK)

  • Integrate transcriptional regulatory networks downstream of TNFRSF17 activation

  • Create multi-scale models connecting molecular events to cellular behaviors

• Multi-omics data integration:

  • Correlate transcriptomic, proteomic, and phosphoproteomic data following TNFRSF17 stimulation

  • Perform time-course analyses to capture network dynamics

  • Identify feedback loops and regulatory mechanisms

  • Apply machine learning to predict network responses to perturbation

• Comparative network analysis:

  • Compare TNFRSF17 signaling networks across:

    • Normal vs. malignant B cells

    • Different B cell developmental stages

    • Various autoimmune conditions

    • Treatment-responsive vs. resistant states

• Perturbation biology approaches:

  • Systematic CRISPR screens targeting TNFRSF17 network components

  • Small molecule inhibitor panels to probe network vulnerabilities

  • Combinatorial perturbations to identify synthetic interactions

  • Single-cell readouts to capture heterogeneous responses

This systems approach has already yielded important insights. For instance, analysis of lupus nephritis identified TNFRSF17 as part of a signature of 70 LN-specific genes significantly enriched in the "regulation of biological quality" GO term and cell cycle pathway . Similarly, in childhood idiopathic nephrotic syndrome, systems analysis revealed that a B cell transcriptional program poised for effector functions, with TNFRSF17 as a key component, represents the major immune perturbation .

By applying these approaches, researchers can move beyond studying TNFRSF17 in isolation to understand how it functions within the complex regulatory landscape of B cells in both health and disease, potentially identifying novel therapeutic targets and biomarkers.