TNFR2 Human Fc

What is TNFR2 and why is it important in immunological research?

TNFR2 is a member of the tumor necrosis factor receptor superfamily primarily expressed on specific immune cells, including T lymphocytes, regulatory T cells (Tregs), endothelial cells, and neural cells. Unlike the ubiquitously expressed TNFR1, TNFR2 has no death domain and activates NF-κB via TRAF2 to promote cell growth rather than apoptosis. Its restricted expression pattern and role in Treg expansion and activation make it a promising target for both cancer immunotherapy and autoimmune disease treatment. TNFR2 signaling is particularly important in regulating immune tolerance, as TNFR2+ Tregs represent the most suppressive Treg subpopulation in both normal physiology and disease contexts .

What is the fundamental difference between TNFR1 and TNFR2 signaling?

TNFR1 and TNFR2 have distinct signaling mechanisms and biological outcomes. TNFR1 contains an intracellular death domain that induces cellular apoptosis by activating caspase 8, promoting inflammatory responses. In contrast, TNFR2 lacks this death domain and instead upregulates NF-κB via TRAF2 to promote cell growth and survival. While TNFR1 is expressed by virtually all cell types and primarily mediates pro-inflammatory effects, TNFR2 expression is limited to specific cell populations and exhibits both pro-inflammatory (e.g., co-stimulation of cytotoxic T cells) and anti-inflammatory activities that promote tissue homeostasis and regeneration .

How do TNFR2-Fc fusion proteins function at the molecular level?

TNFR2-Fc fusion proteins typically combine the extracellular domain of TNFR2 with the Fc portion of human IgG1. This structural arrangement serves multiple purposes: it extends serum half-life through FcRn interaction, creates a bivalent molecule that can more effectively engage with TNF ligands, and enables purification through protein A/G affinity. The Fc domain provides structural stability while keeping sufficient distance from the TNFR2 portion to prevent interference with receptor-ligand interactions. In some designs, N297A mutations are introduced in the Fc region to minimize Fcγ receptor interactions while maintaining FcRn binding for improved pharmacokinetics .

What are the optimal expression systems for producing functional TNFR2-Fc fusion proteins?

Mammalian expression systems, particularly Expi293F cells, have proven most effective for producing functional TNFR2-Fc fusion proteins. These systems ensure proper folding and post-translational modifications essential for biological activity. When designing expression vectors, pCAG-based systems have demonstrated reliable expression. Researchers should consider incorporating appropriate signal peptides for secretion and purification tags (His or Avi tags) strategically placed to avoid interference with receptor binding regions. Expression yields vary significantly based on construct design – conventional antibody-based formats typically yield 15-50 μg/ml, while more complex scFv domain variants may produce only 4-17 μg/ml. For optimal results, stable cell line development rather than transient transfection is recommended for large-scale production of consistent TNFR2-Fc fusion proteins .

How can researchers assess the binding affinity and specificity of TNFR2-Fc constructs?

Multiple complementary approaches should be employed to thoroughly characterize TNFR2-Fc constructs. ELISA-based methods using recombinant TNFα as the capture agent can establish basic binding, while surface plasmon resonance (SPR/Biacore) provides quantitative binding kinetics (kon, koff) and equilibrium dissociation constants (KD). Flow cytometry using cells expressing TNFR2 confirms binding in a cellular context. Species cross-reactivity should be systematically evaluated against human, cynomolgus, mouse, and rat TNFR2 to inform future in vivo study designs. Competition assays with TNFα help determine whether the fusion protein competes for the same binding site. Non-denaturing SDS-PAGE can be used to detect the formation of higher-order complexes between TNFR2-Fc and its target receptor, as evidenced by the appearance of upper-band shifts compared to TNFR2 protein alone .

What functional assays are most informative for evaluating TNFR2-Fc activity in vitro?

For antagonistic TNFR2-Fc constructs, key assays include: (1) TNFα-induced cell death inhibition in TNFR2-overexpressing Jurkat cells, which tests the ability to block TNFα/TNFR2 interactions; (2) assessment of Treg proliferation inhibition using isolated human Tregs labeled with proliferation dyes; and (3) measurement of NF-κB pathway activation. For agonistic constructs, essential assays include: (1) TNFR2/Fas-preadipocyte viability assays that demonstrate concentration-dependent signaling through chimeric receptors; (2) Treg expansion assays showing CD4+CD25+FOXP3+ cell proliferation; and (3) functional suppression assays measuring the ability of TNFR2-stimulated Tregs to inhibit conventional T cell proliferation. Cell-based reporter assays using NF-κB response elements driving luciferase or GFP expression provide quantitative measurements of signaling pathway activation and should include appropriate controls to distinguish between agonistic and antagonistic activities .

How does the domain architecture affect the agonistic activity of TNFR2-targeting molecules?

Domain architecture is the pivotal factor enabling FcγR-independent intrinsic TNFR2 agonism. Two key structural principles have emerged: (1) increasing the valency of unidirectionally aligned TNFR2-binding sites, and (2) creating cell-cell connecting capacity. Anti-TNFR2 antibody formats with either TNFR2 binding sites on opposing sides of the antibody scaffold or those containing six or more TNFR2 binding sites in similar orientation consistently demonstrate strong FcγR-independent agonism. Single-chain variable fragment (scFv) domains connected N-terminally to the CH1 and CL domains via peptide linkers provide sufficient flexibility for binding to different TNFR2+ cells, triggering spontaneous clustering of TNFR2-construct complexes in cell-to-cell contact zones. Interestingly, the affinity of the TNFR2 binding domain and the specific epitope recognized on TNFR2 appear to be secondary factors compared to domain architecture in determining agonistic activity .

How can researchers distinguish between FcγR-dependent and intrinsic TNFR2 agonism?

Distinguishing between FcγR-dependent and intrinsic TNFR2 agonism requires systematic comparative testing. First, researchers should perform parallel experiments with wild-type Fc and N297A-mutated Fc constructs, as the latter prevents FcγR interaction. Second, activity should be assessed in the presence and absence of FcγR-expressing cells using purified Tregs or reconstituted systems. Third, cell-based assays should be conducted with and without anti-Fc crosslinking antibodies to determine if crosslinking enhances activity. Fourth, molecular clustering can be visualized using techniques like proximity ligation assay (PLA) or fluorescence resonance energy transfer (FRET) to assess receptor oligomerization patterns. Finally, dose-response curves in these various conditions provide quantitative evidence of FcγR-dependency. True intrinsic agonists will show concentration-dependent activity regardless of FcγR availability or crosslinking, while FcγR-dependent agonists will show significantly reduced activity in conditions lacking these factors .

What experimental models best demonstrate the efficacy of TNFR2-Fc fusion proteins in autoimmune diseases?

Multiple experimental models have effectively demonstrated the efficacy of TNFR2-Fc fusion proteins in autoimmune diseases. For skin-related autoimmunity, the contact hypersensitivity (CHS) model shows how TNFR2 agonists can reduce ear swelling and inflammatory infiltrates. Collagen-induced arthritis (CIA) models provide evidence for reduced joint inflammation and bone destruction following TNFR2 agonist treatment. Experimental autoimmune encephalomyelitis (EAE) models demonstrate how TNFR2 agonists can attenuate central nervous system inflammation and improve motor function. Graft-versus-host disease (GvHD) models show reduced mortality and tissue damage with TNFR2 agonist therapy. Each model should include comprehensive immunophenotyping of Tregs (measuring numbers, activation markers like CD39, CTLA-4, and suppressive activity) to correlate with disease outcomes. For maximum translational relevance, researchers should consider humanized models that reflect human TNFR2 biology, as species differences in TNFR2 signaling can affect therapeutic outcomes .

How does a single dose of TNFR2 agonist affect Treg populations in vivo?

A single injection of optimized TNFR2 agonists like NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) produces remarkable expansion of regulatory T cells in vivo. Studies demonstrate that Treg numbers increase by more than 300% five days after treatment and remain elevated at approximately 200% for about ten days thereafter. Beyond numerical expansion, TNFR2 stimulation enhances Treg functionality by upregulating the adenosine-regulating ectoenzyme CD39 and other activation markers. These TNFR2-stimulated Tregs demonstrate superior suppressive capacity compared to unstimulated Tregs, more effectively reducing conventional T cell proliferation and expression of activation markers in vitro. The prolonged effect from a single dose highlights the importance of optimal construct design for in vivo stability. This significant expansion and functional enhancement with a single dose demonstrates the potent immunomodulatory potential of well-designed TNFR2 agonists and suggests that intermittent dosing regimens might be sufficient for therapeutic efficacy in autoimmune conditions .

How can researchers address the species specificity issues when developing TNFR2-Fc constructs?

Addressing species specificity challenges requires a multi-faceted approach. First, researchers should systematically test binding to TNFR2 from multiple species (human, cynomolgus, mouse, rat) during early development to identify cross-reactivity. When developing human TNFR2-specific constructs that lack murine cross-reactivity (like AN3025), researchers can employ TNFR2-humanized mouse models for in vivo studies. Another approach involves developing species-specific variants in parallel (e.g., R2agoTNF for mice and R2-7 for humans). For broader applicability, researchers can focus development efforts on the conserved regions of TNFR2 across species. When designing antibody-based TNFR2 targeting molecules, incorporating the variable domains from antibodies with known cross-reactivity can help overcome species barriers. Finally, careful epitope mapping to identify conserved binding regions can guide rational design of constructs with broader species reactivity. Each approach has trade-offs between maintaining specific functional properties and achieving cross-species activity .

What analytical methods are most effective for characterizing the oligomeric state of TNFR2-Fc fusion proteins?

A comprehensive analytical toolkit is necessary for thorough characterization of TNFR2-Fc fusion protein oligomeric states. Size exclusion chromatography (SEC) paired with multi-angle light scattering (MALS) provides accurate molecular weight determination in solution. Analytical ultracentrifugation (AUC) offers complementary data on sedimentation properties related to size and shape. Non-denaturing (native) PAGE can reveal distinct oligomeric forms, as demonstrated by the detection of higher-order complexes between TNFR2-Fc and recombinant TNFR2 protein. Negative-stain transmission electron microscopy (TEM) enables direct visualization of oligomeric structures. Dynamic light scattering (DLS) provides information about the hydrodynamic radius and polydispersity. Cross-linking mass spectrometry can identify interaction interfaces within oligomeric complexes. Finally, small-angle X-ray scattering (SAXS) generates low-resolution structural models of the oligomeric assemblies in solution. These complementary approaches together provide a comprehensive view of the oligomeric state and structural organization of TNFR2-Fc fusion proteins .

How can researchers mitigate the potential immunogenicity of TNFR2-Fc fusion proteins?

Mitigating immunogenicity requires attention to multiple design aspects. First, humanization of non-human components through CDR grafting (as demonstrated with AN3025) significantly reduces immunogenicity. Second, avoiding unnecessary linker sequences or using naturally occurring human protein sequences for linkers can minimize neo-epitope formation. Third, in silico prediction tools should be employed to identify and modify potential T-cell epitopes in the construct. Fourth, deimmunization strategies to remove known MHC-II binding motifs while preserving function can be applied. Fifth, strategic glycoengineering to optimize glycosylation patterns can reduce immunogenic potential. Sixth, careful purification to eliminate protein aggregates, which are highly immunogenic, is essential. For preclinical assessment, researchers should conduct in vitro T cell proliferation assays using human PBMCs and ex vivo immunogenicity studies in humanized mouse models. Testing multiple construct variants in parallel allows selection of the least immunogenic candidate with retained functionality .

What are the key considerations when interpreting conflicting TNFR2 signaling data?

When faced with conflicting TNFR2 signaling data, several factors require careful consideration. First, the context-dependent nature of TNFR2 signaling means results may vary based on cell type, activation state, and microenvironment. Second, different TNFR2 constructs (antagonistic versus agonistic) can yield opposite effects—hence, molecular characterization of the specific construct is essential for proper interpretation. Third, differences in oligomeric state and valency significantly affect signaling outcomes; hexameric or higher-order structures typically demonstrate stronger agonism than trimeric forms. Fourth, species differences in TNFR2 biology can lead to discrepant results between human and murine systems. Fifth, the presence or absence of FcγR-expressing cells in experimental systems dramatically affects results with certain constructs. Sixth, crosslinking conditions vary between studies and must be standardized. Finally, downstream readouts (NF-κB activation, gene expression, cell proliferation) may reflect different aspects of TNFR2 signaling, requiring integrated analysis across multiple parameters for comprehensive understanding .

How should researchers design comparative studies between TNFR2-Fc fusion proteins and anti-TNFR2 antibodies?

Designing robust comparative studies requires systematic methodology. First, establish equivalent molar concentrations rather than mass-based dosing to account for different molecular weights. Second, include positive and negative controls with defined agonistic or antagonistic properties. Third, implement a dose-response matrix covering at least three orders of magnitude concentration range to accurately determine EC50/IC50 values. Fourth, utilize multiple cell types including primary Tregs, conventional T cells, and reporter cell lines to assess context-dependent effects. Fifth, incorporate both short-term (signaling) and long-term (proliferation, gene expression) readouts. Sixth, perform head-to-head comparison in both FcγR-dependent and independent conditions. Seventh, assess the kinetics of receptor binding, internalization, and downstream signaling. Finally, compare constructs in relevant in vivo models where disease-specific outcomes and immune cell profiling provide functional context. This comprehensive approach enables identification of critical performance differences between construct classes and guides rational selection for specific therapeutic applications .

What are the recommended approaches for studying the intracellular signaling cascades activated by TNFR2-Fc constructs?

A multi-layered approach is essential for comprehensive characterization of TNFR2 intracellular signaling. At the protein level, Western blotting to detect phosphorylation of key signaling components (TRAF2, NF-κB p65, IκB) at multiple time points (5-120 minutes) provides dynamic signaling profiles. Phospho-flow cytometry offers single-cell resolution to assess signaling heterogeneity within populations. For pathway mapping, selective inhibitors of NF-κB, MAPK, PI3K, and other pathways help delineate signaling dependencies. At the transcriptional level, time-course RNA-seq analysis captures the temporal dynamics of gene expression changes, while ChIP-seq for NF-κB identifies direct transcriptional targets. Functional validation using CRISPR-mediated knockout of pathway components confirms their necessity. For spatial organization, confocal microscopy tracking receptor clustering and co-localization with signaling components reveals assembly dynamics. Finally, systems biology approaches integrating these datasets can identify signaling nodes and feedback mechanisms. This comprehensive characterization enables precise understanding of how different TNFR2-Fc constructs may differentially activate signaling pathways leading to distinct biological outcomes .

What novel TNFR2-Fc fusion designs might overcome current limitations in specificity and activity?

Emerging research suggests several innovative design approaches that could address current limitations. First, bispecific TNFR2-Fc constructs that simultaneously target TNFR2 and a cell-type specific marker (CD25 for Tregs or tumor-associated antigens for cancer cells) could enhance targeting precision. Second, switchable TNFR2 agonists incorporating small molecule-dependent dimerization domains would enable temporal control of activity. Third, TNFR2-Fc constructs with engineered pH-dependent binding could selectively function in specific tissue microenvironments (e.g., inflammatory or tumor sites). Fourth, incorporating computationally designed de novo binding domains optimized specifically for TNFR2 could overcome the limitations of naturally derived binding domains. Fifth, combining multiple TNF superfamily receptor specificities (e.g., TNFR2/OX40 or TNFR2/4-1BB) in a single molecule could synergistically enhance desired immune responses. Finally, biomaterial-conjugated TNFR2-Fc that provides sustained local delivery could improve tissue-specific effects while minimizing systemic exposure. These next-generation approaches represent promising directions for overcoming the specificity, potency, and context-dependency limitations of current TNFR2-Fc designs .

How might TNFR2-Fc constructs be utilized in combination therapy approaches?

TNFR2-Fc constructs offer versatile opportunities for rational combination therapies. For cancer immunotherapy, antagonistic TNFR2-Fc constructs show promise when combined with checkpoint inhibitors like anti-PD-1 antibodies, as demonstrated by enhanced anti-tumor activity in preclinical models. For autoimmune conditions, agonistic TNFR2-Fc constructs could complement conventional immunosuppressants, potentially allowing dose reduction and minimizing side effects. In transplantation, combining TNFR2 agonists with rapamycin or calcineurin inhibitors might enhance tolerance induction while reducing required doses of broadly immunosuppressive agents. For inflammatory bowel disease, local delivery of TNFR2 agonists combined with anti-TNFα therapy could simultaneously block inflammatory TNF signals while promoting regulatory T cell expansion. Sequencing is critical—in cancer settings, TNFR2 antagonism might optimally precede checkpoint blockade to first deplete suppressive Tregs before activating effector T cells. Rigorous preclinical testing of various dose ratios, scheduling, and careful monitoring for unexpected antagonistic effects are essential for successful clinical translation of these combination approaches .

What are the implications of tissue-specific TNFR2 expression for targeted therapy development?

The tissue-specific expression pattern of TNFR2 creates both opportunities and challenges for targeted therapy development. Beyond immune cells, TNFR2 is expressed on neurons, endothelial cells, and certain epithelial populations, suggesting potential off-target effects requiring careful consideration. In the nervous system, TNFR2 promotes tissue homeostasis and regeneration—thus, antagonistic approaches for cancer therapy might risk neurological adverse effects. Conversely, TNFR2 agonism for autoimmune disease treatment might provide neuroprotective benefits beyond immunomodulation. Endothelial TNFR2 expression impacts vascular integrity and angiogenesis; therefore, monitoring cardiovascular parameters during TNFR2-targeted therapy is advisable. Developing tissue-selective TNFR2-Fc constructs through incorporation of tissue-specific binding domains or using targeted delivery systems could enhance therapeutic index. Single-cell transcriptomic profiling of TNFR2 expression across tissues in health and disease states will inform more precise targeting strategies. Understanding tissue-specific co-receptors and signaling partners could enable the design of context-selective TNFR2 modulators that preferentially act on specific cell populations while sparing others .