Recombinant Human Tumor necrosis factor receptor superfamily member 9 protein (TNFRSF9),Partial (Active)

What is TNFRSF9 and what is its molecular structure?

TNFRSF9, also known as 4-1BB or CD137, is an inducible co-stimulatory receptor primarily expressed on activated T cells. The mature human TNFRSF9 consists of a 163 amino acid extracellular domain (ECD) with four TNFR cysteine-rich repeats, a 27 amino acid transmembrane segment, and a 42 amino acid cytoplasmic domain . Recombinant forms typically include amino acids Leu24-His183/Gln186 of the native protein, sometimes with additional tags (such as 6xHis or 10xHis) for purification and detection purposes . The protein exists as a disulfide-linked homodimer on cell surfaces with an apparent molecular weight of approximately 28-30 kDa, although recombinant monomeric forms may show lower molecular weights (approximately 21.8 kDa) when analyzed by techniques like SEC-MALS .

What cell types express TNFRSF9 and under what conditions?

TNFRSF9 shows a complex expression pattern that varies by cellular context:

• Primary expression occurs on various populations of activated T cells, including CD4+, CD8+, memory CD8+, NKT cells, and regulatory T cells

• Expression is also found on myeloid and mast cell progenitors, dendritic cells, and bacterially infected osteoblasts

• In human gliomas, a surprising expression pattern has been identified primarily in non-neoplastic reactive astrocytes rather than in classic immunological cell types such as lymphocytes and microglia

• In glioma tissue, TNFRSF9 shows predominantly perivascular and peritumoural distribution, with significantly higher expression observed in IDH-1 mutant gliomas

The inducible nature of TNFRSF9 means its expression is highly dependent on cellular activation state rather than being constitutively present.

How does the TNFRSF9-TNFSF9 signaling axis function?

TNFRSF9 binds with high affinity to its ligand TNFSF9 (4-1BB Ligand), which is primarily expressed on antigen-presenting cells and myeloid progenitor cells . This interaction:

• Costimulates the proliferation, activation, and survival of TNFRSF9-expressing cells

• Plays critical roles in the activation, differentiation, and apoptosis of T cells

• Mediates anti-tumor immune responses through enhanced activity of T cells and NK cells

• May contribute to the pathogenesis of certain autoimmune diseases through excessive immune activation

• Can enhance activation-induced cell death in repetitively stimulated T cells

In functional assays, recombinant TNFRSF9 proteins typically bind to TNFSF9 with EC50 values in the range of 1.011-3.702 ng/mL, indicating a high-affinity interaction .

What are the optimal methods for assessing TNFRSF9-TNFSF9 binding interactions?

Several methodologies can effectively characterize TNFRSF9-TNFSF9 binding:

Functional ELISA:

• Immobilize recombinant TNFRSF9 (typically 50 ng/mL)

• Add varying concentrations of TNFSF9

• Detect binding using secondary detection reagents

• Expected EC50 values: 1.011-3.702 ng/mL for high-quality preparations

Surface Plasmon Resonance (SPR):

• Provides real-time kinetic analysis of binding events

• Can determine association/dissociation rates and binding affinities

• Has been successfully employed in studies characterizing TNFRSF9-targeting bispecific antibodies

Cell-Based Binding Assays:

• Utilizes cells expressing TNFRSF9 and fluorescently-labeled TNFSF9

• Provides information about binding in a cellular context

• Can be analyzed by flow cytometry

The choice of method depends on specific research questions - ELISA is suitable for routine binding confirmation, while SPR provides more detailed binding kinetics information essential for therapeutic development.

What are the critical quality attributes for recombinant TNFRSF9 proteins?

When evaluating recombinant TNFRSF9 proteins for research applications, several critical quality attributes should be assessed:

Variations from expected specifications may indicate issues with protein folding, stability, or post-translational modifications that could affect experimental outcomes.

How should researchers handle carrier-free versus carrier-containing preparations of TNFRSF9?

The choice between carrier-free and carrier-containing preparations involves important methodological considerations:

Carrier-Free Preparations:

• Recommended for applications where BSA or other carriers might interfere

• Typically lyophilized from PBS solutions and should be reconstituted at higher concentrations (approximately 500 μg/mL)

• More susceptible to adsorption losses and stability issues

• Requires careful handling to prevent protein loss during dilution and storage

• Essential for certain binding assays, SPR studies, and in vivo applications

Carrier-Containing Preparations:

• Include stabilizers like BSA that enhance shelf-life and stability

• Allow for storage at more dilute concentrations

• More resistant to freeze-thaw cycles and handling stresses

• Suitable for most cell culture applications and as ELISA standards

• May interfere with certain downstream applications or introduce unwanted variables

For either format, it is advisable to avoid repeated freeze-thaw cycles by preparing single-use aliquots and storing at -80°C for maximum retention of activity.

How is TNFRSF9 utilized in CAR-T cell engineering and what are the design considerations?

TNFRSF9 is widely implemented in second-generation CAR-T cell therapy as a costimulatory domain, with several key design considerations:

Domain Selection:

• The intracellular domain of TNFRSF9 provides critical costimulatory signals that enhance T cell activation and persistence

• Typically, amino acids from the cytoplasmic tail are incorporated into the CAR construct downstream of the CD3ζ signaling domain

Comparative Advantages:

• TNFRSF9-containing CARs typically demonstrate enhanced T cell persistence compared to CD28-based designs

• These CARs show improved memory T cell formation

• May provide better long-term anti-tumor responses, particularly important for solid tumors

Optimization Strategies:

To assess CAR-T cells incorporating TNFRSF9 domains, researchers should evaluate not only immediate cytolytic activity but also long-term persistence, memory formation, and resistance to exhaustion for a complete functional profile.

What approaches are being developed for TNFRSF9-targeted cancer immunotherapy?

TNFRSF9-targeted approaches for cancer immunotherapy include several promising strategies:

Agonistic Antibodies:

• Direct TNFRSF9 agonists that stimulate T cell activation and proliferation

• Have shown remarkable efficacy in murine tumor models but face challenges with systemic toxicity in clinical translation

Bispecific Antibodies:

• Combine TNFRSF9 agonism with targeting of tumor antigens or checkpoint molecules

• Example: CD137×PD-L1 bispecific antibodies that provide context-dependent T cell costimulation and checkpoint blockade

• These designs restrict TNFRSF9 activation to the tumor microenvironment, potentially improving safety

Engineered TNFSF9 Ligands:

• Recombinant versions of the natural ligand with enhanced stability or targeting properties

• May provide more physiological receptor activation compared to antibody approaches

Methodological Considerations:

• Careful epitope selection significantly impacts agonistic activity

• Antibody format affects pharmacokinetics and tissue penetration

• Fc engineering can modulate FcγR binding to enhance agonistic activity

• Affinity balancing is critical to prevent systemic activation and toxicity

Clinical development has been challenging due to hepatotoxicity observed with some TNFRSF9 agonists, highlighting the need for more selective targeting approaches.

What is the significance of TNFRSF9 upregulation in gliomas?

Research has revealed a unique pattern of TNFRSF9 expression in human gliomas with important implications:

Expression Pattern:

• TNFRSF9 is considerably upregulated in human gliomas compared to normal brain tissue

• Surprisingly, expression is predominantly found in non-neoplastic reactive astrocytes rather than in tumor cells or typical immune cells

• Distribution is primarily perivascular and peritumoural, with significantly higher expression in IDH-1 mutant gliomas

Research Implications:

• This pattern challenges direct translation of findings from murine models where TNFRSF9-targeting approaches have shown complete tumor eradication

• Identifies a novel TNFRSF9-positive reactive astrocytic phenotype that may have unique functional properties in the tumor microenvironment

• Suggests TNFRSF9 might be involved in forming a reactive tumor microenvironment rather than direct anti-tumor immunity in this context

Methodological Recommendations:

• Combined immunohistochemistry and immunofluorescence approaches with cell-type specific markers are essential for accurate characterization

• Careful validation of antibody specificity is critical given the unexpected cellular localization

• Single-cell analysis may provide further insights into heterogeneity of expression within reactive astrocyte populations

These findings highlight the importance of thorough characterization of target expression patterns in human samples before clinical translation of promising preclinical results.

How is TNFRSF9 expression regulated at the epigenetic level?

Emerging research has begun to elucidate the epigenetic regulation of TNFRSF9:

DNA Methylation:

• Multiple CpG sites within the TNFRSF9 gene show differential methylation patterns that correlate with expression levels

• At least twelve CpG sites have been identified as potentially relevant for regulation

• Methylation patterns may correlate with response to immunotherapy, particularly checkpoint inhibitors in melanoma

Methodological Approaches:

• Bisulfite sequencing provides single-nucleotide resolution of methylation status across the TNFRSF9 gene

• Methylation-specific PCR can rapidly assess methylation at specific CpG sites

• Pyrosequencing offers quantitative assessment of methylation levels

• Methylation arrays allow for genome-wide analysis including the TNFRSF9 locus

Research Implications:

• Correlation analyses between methylation patterns and gene expression are critical to establish functional significance

• Integration with clinical outcome data may identify predictive biomarker signatures

• Combined analysis with other epigenetic marks (histone modifications, chromatin accessibility) provides a more complete regulatory picture

Understanding epigenetic regulation of TNFRSF9 may lead to novel therapeutic strategies combining epigenetic modifiers with immunotherapeutic approaches.

What is the potential for TNFRSF9 as a biomarker in immunotherapy?

TNFRSF9 has emerging potential as a biomarker in several contexts:

Predictive Biomarker for Immunotherapy:

• DNA methylation patterns at the TNFRSF9 locus may correlate with response to anti-PD-1 immunotherapy in melanoma

• Expression levels on tumor-infiltrating lymphocytes may indicate immune activation status

• Soluble forms of TNFRSF9 are elevated in certain disease states and may serve as liquid biopsy markers

Prognostic Biomarker:

• Expression patterns in tumor microenvironments may correlate with clinical outcomes

• The distribution pattern (perivascular/peritumoural) appears to have biological significance in gliomas

• Higher expression in IDH-1 mutant gliomas suggests correlation with molecular subtypes

Methodological Considerations:

• Standardized assays with validated cutoff values are needed for clinical application

• Tissue-based versus circulating biomarker approaches have different strengths and limitations

• Integration with other immune markers may provide more robust predictive signatures

• Sample collection timing relative to treatment may significantly impact biomarker performance

Further research correlating TNFRSF9 methylation, expression, and clinical outcomes across multiple cancer types is needed to fully establish its biomarker utility.

What are the emerging approaches for addressing toxicity challenges in TNFRSF9-targeted therapies?

Several innovative strategies are being developed to mitigate the hepatotoxicity and other adverse effects associated with TNFRSF9 agonism:

Conditional Activation Approaches:

• Tumor-targeted bispecific antibodies that activate TNFRSF9 only in the presence of specific tumor antigens

• pH-sensitive antibodies that preferentially activate in the acidic tumor microenvironment

• Protease-activated antibodies that become functional only in the presence of tumor-associated proteases

Engineering Strategies:

• Fc domain modifications that fine-tune FcγR interactions and subsequent agonistic activity

• Alternative antibody formats with optimized valency and binding properties

• Antibody fragments with limited systemic exposure but retained tumor penetration

Combination Approaches:

• Lower-dose TNFRSF9 agonism combined with complementary immunotherapies

• Sequential rather than concurrent administration protocols

• Local rather than systemic administration for specific tumor types

These approaches require sophisticated preclinical models, including humanized mouse models and ex vivo human tissue systems, to accurately predict both efficacy and toxicity before clinical translation.

How might single-cell technologies advance understanding of TNFRSF9 biology?

Single-cell technologies offer unprecedented opportunities to resolve TNFRSF9 biology in complex tissues:

Single-cell RNA Sequencing:

• Reveals cell type-specific expression patterns of TNFRSF9 and TNFSF9

• Identifies co-expressed receptors and downstream signaling components

• Characterizes heterogeneity in expression levels within nominally similar cell populations

CITE-seq and Protein Analysis:

• Simultaneously assesses TNFRSF9 protein levels and transcriptional profiles

• Correlates protein expression with activation states and functional markers

• Identifies post-transcriptional regulatory mechanisms

Spatial Transcriptomics:

• Maps TNFRSF9 expression within tissue architecture

• Correlates expression with specific microenvironmental niches

• Particularly valuable for understanding the perivascular/peritumoural distribution observed in gliomas

Epigenetic Analysis at Single-cell Level:

• Single-cell ATAC-seq reveals chromatin accessibility at the TNFRSF9 locus

• Single-cell methylation analysis identifies cell-specific regulatory patterns

• Integrative multi-omic approaches connect epigenetic state to expression and function

These technologies could resolve contradictory findings between animal models and human samples, such as the unexpected expression of TNFRSF9 on reactive astrocytes in human gliomas, potentially leading to more precisely targeted therapeutic strategies.