Recombinant Human Tumor Necrosis Factor-alpha/TNFSF2 (TNF), partial (Active) (GMP)

What is the molecular structure of Recombinant Human TNF-alpha?

Recombinant Human TNF-alpha is a homotrimeric protein with a molecular weight of approximately 53.1 kDa as determined by size-exclusion chromatography with multi-angle light scattering (SEC-MALS). The protein is derived from E. coli expression systems and spans amino acids Val77-Leu233 of the native sequence, sometimes with an N-terminal methionine. When analyzed by SDS-PAGE under reducing conditions, it appears as a 17 kDa band, representing the monomeric form .

The native human TNF-alpha protein consists of a 35 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 177 amino acid extracellular domain (ECD). Within the ECD, human TNF-alpha shares 97% amino acid sequence identity with rhesus and between 71-92% with other mammalian species including bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-alpha .

For GMP-grade preparations, MALDI-TOF analysis confirms the expected molecular mass of 17,348 Da for the protein without N-terminal Met, and 17,480 Da for the variant with N-terminal Met .

How does TNF-alpha function at the cellular level?

TNF-alpha is a pleiotropic cytokine central to inflammation, immune system development, apoptosis, and lipid metabolism. It is produced by various cell types including immune cells, epithelial cells, endothelial cells, and tumor cells. The protein is initially assembled intracellularly as a noncovalently linked homotrimer and expressed on the cell surface .

At the functional level, TNF-alpha operates through two main mechanisms:

• Membrane-bound form: Cell surface TNF-alpha can induce lysis of neighboring tumor cells and virus-infected cells, and can generate its own downstream signaling following interaction with soluble TNF receptor I (TNFR I) .

• Soluble form: Following cleavage from the cell surface by TACE/ADAM17, the soluble 55 kDa trimeric form is released as the bioactive cytokine that can act on distant cells .

The bioactivity of TNF-alpha is typically measured through cytotoxicity assays using the L-929 mouse fibroblast cell line in the presence of actinomycin D. The ED50 for this effect is consistently in the range of 25-100 pg/mL, demonstrating the potent biological activity of the recombinant protein .

What is the difference between GMP-grade and research-grade Recombinant Human TNF-alpha?

GMP-grade (Good Manufacturing Practice) Recombinant Human TNF-alpha differs from research-grade in several critical aspects:

• Manufacturing conditions: GMP-grade protein is manufactured and tested under cGMP guidelines, ensuring consistent quality and purity for potential therapeutic applications .

• Production environment: GMP-grade TNF-alpha is produced using non-animal reagents in an animal-free laboratory, reducing the risk of contamination with animal-derived pathogens .

• Standardization: The specific activity of GMP-grade human TNF-alpha is calibrated against the human TNF-alpha WHO International Standard (NIBSC code: 12/154) and is typically >4.3 x 10^7 IU/mg .

• Documentation: GMP products are accompanied by more extensive documentation regarding the manufacturing process, quality control, and batch consistency .

• Intended use: While research-grade is intended for in vitro or preclinical research, GMP-grade material is suitable for clinical research and therapeutic development pathways .

Both preparations maintain similar biological activities, with the ED50 for cytotoxicity in L-929 cells remaining in the range of 25-100 pg/mL .

How should Recombinant Human TNF-alpha be reconstituted and stored for optimal activity?

Proper reconstitution and storage are crucial for maintaining the bioactivity of Recombinant Human TNF-alpha. Based on manufacturer guidelines and research protocols, the following methodology is recommended:

Reconstitution Procedure:

• Allow the lyophilized protein to reach room temperature before opening.

• Reconstitute using sterile PBS or other appropriate buffers as specified in the product datasheet.

• Gently agitate until completely dissolved; avoid vigorous shaking which can cause protein denaturation.

• For GMP-grade material, use only sterile, endotoxin-free solutions and materials during reconstitution .

Storage Recommendations:

• Short-term storage (up to 1 month): 2-8°C

• Long-term storage: Aliquot and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles which can reduce bioactivity

• Working solutions should be used within 24 hours when stored at 2-8°C

The bioactivity of properly reconstituted and stored TNF-alpha can be verified through cytotoxicity assays using L-929 cells, with expected ED50 values of 25-100 pg/mL .

What assays are recommended for verifying TNF-alpha bioactivity in research applications?

Several assays are commonly used to verify the bioactivity of Recombinant Human TNF-alpha in research applications:

1. L-929 Cytotoxicity Assay:
This is the gold standard for TNF-alpha bioactivity assessment. The assay measures TNF-alpha-induced cell death in the L-929 mouse fibroblast cell line in the presence of actinomycin D.

2. NF-κB Reporter Assays:
These assays use cells transfected with an NF-κB-responsive reporter gene to measure TNF-alpha-induced signaling activation.

3. Cytokine Induction Assays:
Measuring the production of downstream cytokines (IL-6, IL-8, etc.) in responsive cell types after TNF-alpha stimulation.

4. Upregulation of Surface Markers:
Flow cytometry analysis of TNF-alpha-induced upregulation of cell surface molecules like adhesion molecules (VCAM-1, ICAM-1) on endothelial cells .

5. SOCS1 Expression Assay:
Measuring the upregulation of SOCS1 (Suppressor of Cytokine Signaling 1) gene expression in human peripheral blood mononuclear cells (PBMCs) or isolated islets after exposure to TNF-alpha .

For research reporting, it is recommended to include both positive controls (known bioactive TNF-alpha) and negative controls (heat-inactivated TNF-alpha or buffer-only) to validate assay performance.

What concentrations of TNF-alpha are typically used in in vitro experimental systems?

The appropriate concentration of TNF-alpha varies significantly depending on the experimental system, cell type, and research objective. Based on published research using Recombinant Human TNF-alpha, the following concentration guidelines are recommended:

When designing dose-response experiments, a logarithmic concentration series (e.g., 0.1, 1, 10, 100 ng/mL) is typically recommended to capture the full response range .

It is important to note that cells may exhibit differential sensitivity to TNF-alpha based on receptor expression levels, culture conditions, and cell activation status. Therefore, preliminary dose-finding experiments are advisable when establishing a new experimental system .

How can Recombinant Human TNF-alpha be used in infectious disease research models?

TNF-alpha plays critical roles in infectious disease pathogenesis, particularly in models of tuberculosis and other chronic infections. Research applications include:

Tuberculosis Models:
TNF-alpha is essential for protective immune responses against Mycobacterium tuberculosis by promoting granuloma formation and maintenance. Studies have demonstrated that TNF-alpha synergizes with other cytokines to control both local accumulation and dissemination of pathogens .

Experimental approaches include:

• In vitro infection models: Using TNF-alpha to stimulate macrophages infected with M. tuberculosis to study antimicrobial responses

• Ex vivo granuloma models: Applying TNF-alpha to study granuloma formation and maintenance

• TNF-alpha neutralization studies: Using anti-TNF-alpha antibodies to model reactivation of latent TB infection

Researchers investigating the dual role of TNF-alpha in infection should consider:

• Using physiologically relevant concentrations

• Examining both protective and pathological effects

• Studying interactions with other cytokines and immune factors

• Comparing results across different model systems (human cells, animal models)

What are the applications of TNF-alpha in cancer research and therapeutic development?

Recombinant Human TNF-alpha has significant applications in cancer research, both for understanding tumor biology and developing therapeutic approaches:

Research Applications:

• Direct cytotoxicity studies: TNF-alpha can induce apoptosis in certain tumor cell lines, making it valuable for studying tumor cell death mechanisms.

• Tumor microenvironment modeling: TNF-alpha plays a role in shaping the inflammatory tumor microenvironment and can be used to study tumor-stromal interactions.

• Resistance mechanisms: Many tumors develop resistance to TNF-alpha-induced cell death, providing a model to study apoptosis resistance.

Therapeutic Development:
Modified versions of TNF-alpha have been developed to enhance therapeutic efficacy. In China, a recombinant mutant human TNF (rmhTNF) was developed by modifying the TNF gene using PCR technology, resulting in a non-glycosylated single chain of 151 amino acids with enhanced anti-tumor activity .

This modified rmhTNF features:

• Deletion of the first seven amino acids

• Substitution of four amino acids (Arg for Pro at position 8, Lys for Ser at position 9, Arg for Asp at position 10, and Phe for Leu at position 157)

• Enhanced anti-tumor activity compared to wild-type TNF-alpha

Clinical applications include thoracic perfusion of rmhTNF combined with cisplatin for the treatment of malignant pleural effusion (MPE) in lung cancer patients. Studies have shown higher objective response rates compared to cisplatin alone, suggesting that rmhTNF significantly enhances therapeutic efficacy .

Other TNF-alpha modifications being investigated include NGR (asparagine glycine arginine)-modified TNF-alpha for targeting colorectal cancer, liver cancer, and malignant pleural mesothelioma .

How does Recombinant Human TNF-alpha contribute to diabetes research models?

TNF-alpha plays a complex role in diabetes pathogenesis, and recombinant TNF-alpha is used in various experimental approaches to study both type 1 and type 2 diabetes:

Type 1 Diabetes Models:
Research with streptozotocin (STZ)-induced diabetes in mice has shown that recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is related to TNF-alpha, can ameliorate the severity of diabetes. In one study, mice co-injected with STZ and recombinant TRAIL (20 μg/day for 5 days) showed:

• Lower levels of hyperglycemia

• Higher levels of body weight and insulinemia

• Partial preservation of pancreatic islets with normal morphology

• Lower expression of inflammatory markers (TNF-alpha, osteoprotegerin, VCAM-1)

The mechanism appears to involve upregulation of SOCS1 (Suppressor of Cytokine Signaling 1) expression. In vitro exposure of both human PBMCs and isolated human islets to recombinant TRAIL significantly upregulated SOCS1 expression .

Experimental Protocol for STZ-Diabetes Model:

• Induce diabetes with five consecutive daily injections of low-concentration (50 mg/kg) streptozotocin in mice

• Co-inject experimental group with recombinant TRAIL (20 μg/day) for 5 days

• Monitor diabetic status (glycemia and body weight) over time

• After 6 weeks, measure circulating levels of insulin, TNF-alpha, and osteoprotegerin

• Perform histological analysis of the pancreas

This model provides a valuable approach for studying immunomodulatory interventions in type 1 diabetes and highlights the potential therapeutic value of TNF family proteins in autoimmune diabetes .

How can variable responses to TNF-alpha across different experimental systems be addressed?

Researchers often encounter variability in TNF-alpha responses across different experimental systems. This variability can be addressed through systematic troubleshooting and experimental design considerations:

Common Sources of Variability:

• Receptor Expression: Different cell types express varying levels of TNF receptor I (55-60 kDa) and TNF receptor II (80 kDa). While both receptor types bind TNF-alpha with comparable affinity, only TNF RI contains a cytoplasmic death domain that triggers apoptosis .

• Soluble Receptors: Soluble forms of both receptor types can be released by cells and neutralize TNF-alpha bioactivity, potentially causing inconsistent results in long-term cultures .

• Species Differences: Despite sequence homology between human and mouse TNF-alpha (71-92%), there can be species-specific responses .

Methodological Solutions:

• Receptor Profiling: Before experiments, characterize the expression of TNF-RI and TNF-RII on target cells using flow cytometry or Western blotting.

• Fresh Media Controls: Replace culture media before TNF-alpha treatment to remove accumulated soluble receptors.

• Positive Controls: Include a well-characterized cell line (e.g., L-929) as a positive control in each experiment to confirm TNF-alpha bioactivity.

• Dose-Response Analysis: Perform comprehensive dose-response studies (10 pg/mL to 100 ng/mL) for each new cell type or experimental system.

• Actinomycin D Sensitivity: For cytotoxicity assays, optimize actinomycin D concentration, as different cell types may require different concentrations to sensitize them to TNF-alpha-induced apoptosis.

By implementing these approaches, researchers can significantly reduce variability and increase reproducibility in TNF-alpha experiments across different systems .

What are the key considerations when transitioning from in vitro to in vivo TNF-alpha research?

Transitioning from in vitro to in vivo TNF-alpha research requires careful consideration of several factors to ensure successful experimental outcomes:

Dosing Considerations:

• Physiological Relevance: In vitro effective doses (ED50 of 25-100 pg/mL) may not directly translate to in vivo settings due to pharmacokinetics, tissue distribution, and presence of neutralizing factors .

• Administration Route: Different routes (intravenous, intraperitoneal, subcutaneous, or localized delivery) affect TNF-alpha biodistribution and activity. For example, in diabetes models, direct co-injection with streptozotocin (20 μg/day for 5 days) has been effective .

Systemic vs. Local Effects:

• Systemic Toxicity: TNF-alpha can cause systemic inflammatory responses, fever, and cachexia when administered systemically at high doses.

• Local Delivery Approaches: Consider localized delivery methods such as thoracic perfusion (used for rmhTNF in malignant pleural effusion treatment) to maximize local effects while minimizing systemic toxicity .

Species-Specific Considerations:

• Species Cross-Reactivity: Human TNF-alpha may have different potencies in various animal models. While human TNF-alpha shares 97% amino acid sequence identity with rhesus monkey, it shares only 71-92% with other species .

• Receptor Distribution: The distribution of TNF receptors varies across species and tissues, potentially affecting experimental outcomes.

Monitoring and Safety Parameters:

• Inflammatory Markers: Monitor systemic inflammatory markers (IL-6, CRP) to assess systemic effects.

• Body Weight and Temperature: Regular monitoring of body weight and temperature can provide early indications of TNF-alpha toxicity.

• Organ-Specific Markers: Depending on the disease model, monitor relevant organ-specific markers (e.g., insulin and glucose for diabetes models) .

By carefully addressing these considerations, researchers can design more effective and translatable in vivo experiments using recombinant TNF-alpha .

How are modified versions of TNF-alpha advancing therapeutic applications?

Modified versions of TNF-alpha represent a significant frontier in therapeutic development, addressing limitations of native TNF-alpha while enhancing desirable properties:

Key Modified TNF-alpha Variants:

• Recombinant Mutant Human TNF (rmhTNF):

  • Developed in China and approved by the China State Food and Drug Administration (SFDA) for cancer treatment

  • Features deletion of the first seven amino acids and substitution of four amino acids (Arg for Pro at position 8, Lys for Ser at position 9, Arg for Asp at position 10, and Phe for Leu at position 157)

  • Shows enhanced anti-tumor activity compared to native TNF-alpha

  • Effective in treating malignant pleural effusion when combined with cisplatin, showing higher objective response rates than cisplatin alone

• NGR-Modified TNF-alpha:

  • TNF-alpha modified with asparagine-glycine-arginine (NGR) peptide

  • Targets CD13 (aminopeptidase N) expressed on tumor vasculature

  • Being investigated for treating colorectal cancer, liver cancer, and malignant pleural mesothelioma

  • Provides more targeted delivery to tumor tissue, potentially reducing systemic side effects

• PEGylated TNF-alpha:

  • Addition of polyethylene glycol (PEG) moieties increases half-life and reduces immunogenicity

  • Allows for less frequent dosing and potentially reduced side effects

Research Applications:

These modified TNF-alpha variants enable new research approaches, including:

• Targeted Delivery Studies: Investigating tissue-specific effects when TNF-alpha is delivered to specific organs or cell types

• Combination Therapy Research: Studying synergistic effects with chemotherapy agents (e.g., cisplatin)

• Reduced Toxicity Models: Allowing higher dosing with lower systemic toxicity for better understanding of dose-dependent effects

As these modified forms continue to develop, they provide valuable tools for both therapeutic applications and basic research into TNF-alpha biology and signaling mechanisms .

What role does TNF-alpha play in immunomodulatory research beyond inflammation?

While traditionally viewed primarily as a pro-inflammatory cytokine, research has revealed that TNF-alpha has complex immunomodulatory roles beyond simple inflammation induction:

Immunoregulatory Functions:

• SOCS1 Upregulation: Research has demonstrated that TNF-family cytokines like TRAIL can significantly upregulate SOCS1 (Suppressor of Cytokine Signaling 1) expression in both human peripheral blood mononuclear cells (PBMCs) and isolated human islets. This immunoregulatory mechanism may contribute to anti-inflammatory effects in certain contexts .

• Dual Roles in Infectious Disease: TNF-alpha shows contradictory functions in infectious disease contexts:

  • Essential for protective immunity (e.g., granuloma formation in tuberculosis)

  • Can drive pathology and disease symptoms when produced in excess
    Understanding this balance requires sophisticated experimental approaches

• T Cell Homeostasis: TNF-alpha influences T cell activation, differentiation, and apoptosis, contributing to T cell homeostasis and tolerance mechanisms.

Experimental Approaches for Studying Immunomodulatory Functions:

• Gene Expression Profiling: Measure expression of immunoregulatory genes like SOCS1 after TNF-alpha treatment of immune cells or target tissues .

• Functional Assays in Complex Models: For example, in the streptozotocin-induced diabetes model, co-administration of TRAIL (a TNF-family member) resulted in:

  • Lower hyperglycemia

  • Higher insulinemia

  • Partial preservation of pancreatic islets

  • Reduced inflammatory markers

• Receptor-Specific Studies: Distinguishing between signaling through TNF receptor I (which contains a death domain) versus TNF receptor II (which lacks a death domain) to understand differential immunomodulatory effects .

These research directions highlight the importance of viewing TNF-alpha beyond its classical pro-inflammatory role, opening new possibilities for therapeutic interventions in autoimmune and inflammatory conditions .

What are the emerging techniques for studying TNF-alpha signaling mechanisms?

The study of TNF-alpha signaling is advancing through innovative techniques that provide deeper insights into its complex biology:

Advanced Imaging Techniques:

• Live-cell imaging with fluorescently-tagged TNF-alpha and receptors to track receptor-ligand dynamics in real time

• Super-resolution microscopy to visualize TNF-alpha receptor clustering and signaling platform assembly at the nanoscale level

• Intravital imaging to observe TNF-alpha effects in live tissues within animal models

Genomic and Proteomic Approaches:

• Single-cell RNA sequencing to characterize heterogeneous responses to TNF-alpha across individual cells within a population

• Phosphoproteomics to map TNF-alpha-induced signaling cascades comprehensively

• CRISPR-Cas9 screening to identify novel mediators of TNF-alpha signaling

Biosensor Technologies:
Recent developments include engineered cell lines containing biosensors that provide real-time readouts of TNF-alpha-induced NF-κB activation, allowing for more dynamic studies of signaling kinetics and modulation .

These emerging techniques are enabling researchers to address longstanding questions about the context-dependent effects of TNF-alpha across different tissue and disease settings.

How is TNF-alpha research contributing to precision medicine approaches?

TNF-alpha research is increasingly contributing to precision medicine strategies through several important avenues:

Biomarker Development:

• Predictive biomarkers for anti-TNF therapy response in inflammatory diseases

• Monitoring biomarkers to track disease activity and treatment efficacy

• Risk stratification markers based on TNF pathway genomics and proteomics

Targeted Delivery Approaches:
The development of modified TNF-alpha variants like NGR-modified TNF-alpha that target specific tissues (e.g., tumor vasculature) represents an important advance toward precision medicine approaches that can deliver therapeutic agents to disease sites while sparing healthy tissues .

Personalized Immunotherapy:
Understanding individual variations in TNF-alpha signaling pathways is informing more personalized approaches to immunotherapy, particularly in cancer treatment where rmhTNF has shown promising results in combination with conventional chemotherapy for conditions like malignant pleural effusion .

Disease-Specific Applications:
Research into the specific roles of TNF-alpha in various disease contexts (tuberculosis, diabetes, cancer) is revealing distinct mechanisms that can be targeted in a disease-specific manner, moving away from one-size-fits-all approaches to more nuanced interventions based on specific pathogenic mechanisms .