Elicit: Adalimumab and TNF Receptor Binding
Adalimumab p55 p75 TNF receptor binding
Adalimumab does not directly bind to p55 or p75 TNF receptors; instead, it binds to TNFα and sterically blocks TNFα from engaging these receptors.
Abstract
No studies directly measured adalimumab binding to isolated p55 (TNF-RI) or p75 (TNF-RII) receptors. Instead, adalimumab exerts its effects by binding to TNFα with high affinity (30.4 pM) and sterically blocking TNFα-receptor interactions through epitope overlap with the TNFR2 binding site. The adalimumab epitope comprises discontinuous TNFα segments with a buried surface area of 2,540 Ų, competitively inhibiting TNFα binding to both receptor subtypes and preventing activation of downstream signaling pathways including NF-κB. Functional studies revealed differential effects on receptor preservation: adalimumab at 16.7 nM maintained TNF-RI surface expression at baseline levels while TNF reduced it 2.4-fold, whereas all anti-TNF biologics equally prevented TNF-RII loss. Blocking antibody studies confirmed that TNF-RI is the predominant receptor mediating TNF-dependent cellular responses, which adalimumab inhibits by restricting TNF access to this receptor. These findings indicate that adalimumab does not directly bind p55 or p75 receptors, but rather blocks TNFα from engaging these receptors, with functional selectivity favoring TNF-RI preservation through mechanisms independent of the Fc domain.
Methods
We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question. More on methods.
Paper search
We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.
We ran this query: "Adalimumab p55 p75 TNF receptor binding".
The search returned 200 total results from Elicit.
We retrieved 200 papers most relevant to the query for screening.
Screening
We screened in sources based on their abstracts that met these criteria:
- Adalimumab Binding Mechanisms: Does this study investigate adalimumab’s binding mechanisms or interactions with TNF receptors?
- Specific TNF Receptor Types: Does this study examine TNF-α receptor p55 (TNFR1) and/or p75 (TNFR2) in relation to adalimumab?
- Binding Measurements: Does this study measure binding affinity, receptor occupancy, or molecular interactions?
- Appropriate Study Design: Is this study an in vitro, in vivo, ex vivo experimental study, clinical trial, or pharmacokinetic/pharmacodynamic study?
- Mechanistic Data Inclusion: Does this study include mechanistic data on TNF receptor binding (not solely clinical outcomes)?
- Adalimumab Focus: Does this study focus on adalimumab specifically, or if it studies other TNF inhibitors, does it include direct comparison to adalimumab?
- Study Type Quality: Is this study a full research article (not a case report, editorial, opinion piece, or conference abstract without full-text publication)?
We considered all screening questions together and made a holistic judgement about whether to screen in each paper.
Data extraction
We asked a large language model to extract each data column below from each paper.
TNF Receptor Binding Data:
Extract all binding data for adalimumab to p55 (TNF-RI) and p75 (TNF-RII) TNF receptors, including:- Binding affinity values (KD, IC50, etc.) for p55 and p75 separately
- Association and dissociation rates (kon, koff)
- Avidity measurements
- Binding stoichiometry
- Surface plasmon resonance data
- Competition binding results
- Any quantitative binding parameters specific to these two receptor subtypes
Receptor Expression Effects:
Extract data on how adalimumab affects p55 (TNF-RI) and p75 (TNF-RII) receptor expression or availability, including:- Changes in surface receptor levels
- Receptor internalization or externalization
- Receptor preservation vs. loss in presence of TNF
- Flow cytometry data for receptor expression
- Time-dependent changes in receptor availability
- Dose-dependent effects on receptor expression
Binding Mechanism:
Extract molecular details of adalimumab’s binding mechanism to TNF receptors p55 and p75, including:- Epitope mapping data
- Crystal structure information
- Binding interface characteristics
- Competitive inhibition with natural TNF-receptor binding
- Whether binding blocks TNF access to receptors
- Allosteric effects or conformational changes
- Complex formation details (TNF:adalimumab:receptor)
Functional Consequences:
Extract functional outcomes resulting from adalimumab binding to p55/p75 TNF receptors, including:- Effects on TNF-mediated signaling pathways (NF-kB, etc.)
- Changes in downstream cellular responses
- Apoptosis induction or inhibition
- Cytokine production changes
- Cell surface marker expression (ICAM-1, etc.)
- Any receptor-specific functional differences between p55 vs p75
Experimental Methods:
Extract the experimental approaches used to study adalimumab binding to TNF receptors p55 and p75, including:- Cell lines or primary cells used
- Binding assay techniques (SPR, flow cytometry, radioligand binding)
- TNF receptor source (soluble vs membrane-bound)
- Assay conditions (concentrations, incubation times, temperature)
- Detection methods and equipment
- Controls used
Comparative Analysis:
Extract comparative data between adalimumab and other TNF inhibitors (etanercept, infliximab, golimumab, certolizumab) specifically regarding p55/p75 TNF receptor binding, including:- Head-to-head binding comparisons
- Relative efficacy in blocking receptor binding
- Differences in receptor selectivity (p55 vs p75)
- Comparative functional outcomes
- Any advantages or disadvantages of adalimumab vs other agents
Results
Characteristics of Included Studies
The review included 10 studies examining adalimumab’s interactions with TNF receptors, published between 2009 and 2022. Full text was available for 3 of 10 studies.
Study
| Study | Full Text Retrieved? | Primary Focus | Study Type |
|---|---|---|---|
| Z. Kaymakcalan et al., 2009 | No | Comparative binding properties and complement activation of adalimumab, infliximab, and etanercept | In vitro binding and functional assays |
| Shi Hu et al., 2013 | Yes | Crystal structure of TNFα-adalimumab complex and epitope mapping | Structural analysis and binding studies |
| U. Kronthaler et al., 2018 | No | Biosimilar characterization of GP2017 vs. reference adalimumab | In vitro functional and binding assays |
| B. Harvey & Z. Kaymakcalan, 2014 | No | Mechanism of adalimumab inhibition of TNF-enhanced osteoclast function | Cell-based functional assays with primary human osteoclast precursors |
| Elena Krayukhina et al., 2017 | Yes | Complex formation and FcγR activation by TNF antagonists | Analytical ultracentrifugation and reporter assays |
| Shi Hu et al., 2014 | Yes | Corrected epitope mapping data for adalimumab-TNFα interactions | Structural analysis correction |
| J. Watkins & J. Watkins, 2022 | No | Engineered pH-sensitive adalimumab variant to reduce immunogenicity | In vitro functional assays with THP1 monocytes |
| B. Harvey & Z. Kaymakcalan, 2019 | No | Functional differences between anti-TNF biologics on human monocytes | Cell-based assays with U937 and primary PBMCs |
| S. Halder et al., 2019 | No | Biosimilar ABP 501 characterization supporting IBD extrapolation | Multiple binding and functional assays |
| T. Arora et al., 2009 | No | Comparative binding and effector functions of TNF antagonist classes | In vitro binding and cytotoxicity assays |
Most studies did not provide direct binding data for p55 (TNF-RI) and p75 (TNF-RII) receptors specifically. Instead, studies focused on adalimumab’s binding to soluble and membrane-bound TNFα, with functional consequences on receptor-mediated signaling inferred indirectly. Three studies provided full text access, enabling more detailed structural and mechanistic analysis.
Binding Characteristics and Epitope Mapping
TNFα Binding Affinity and Epitope
Adalimumab demonstrated binding affinity to TNFα with KD values ranging from 7.05 × 10^-11 M to 1.0 × 10^-10 M. Surface plasmon resonance analysis revealed a binding affinity of 30.4 pM. Crystal structure analysis identified the adalimumab epitope on TNFα as comprising discontinuous segments including residues TNF Pro-20, TNF Gln-21, TNF Glu-23, TNF Lys-65 to TNF Gln-67, TNF Glu-110 to TNF Pro-113, TNF Tyr-141, TNF Ala-145 to TNF Glu-146, and TNF Thr-72, TNF His-73, TNF Thr-77, TNF Thr-79, TNF Ser-81, TNF Lys-90 to TNF Asn-92, and TNF Glu-135 to TNF Asn-137 of an adjacent TNFα protomer.
The crystal structure of the TNFα-adalimumab Fab complex revealed a large and highly complementary interface with a total buried surface area of 2,540 Ų. This interface was primarily composed of residues in the G-H loop, several amino acids in strand D, and the D-E loop of an adjacent TNFα protomer. Critically, the adalimumab epitope occupied a larger area compared to infliximab and overlapped substantially with the TNFR binding site, indicating that adalimumab competitively inhibits the binding of TNFR to TNFα by occupying the receptor-binding site. This mechanism of action involves both steric blocking and prevention of ligand binding.
Complex Formation
Analytical ultracentrifugation with fluorescence detection revealed that adalimumab formed a range of complexes with TNF, with major complexes consisting of 3 molecules of adalimumab and one or 2 molecules of TNF. The crystal structure confirmed that complexes consisted of one TNF trimer bound by 3 Fab molecules. Surface plasmon resonance studies using BIAcore T100 employed a contact time of 300 seconds and a flow rate of 30 μL/min.
Effects on TNF Receptor Expression and Function
Receptor Surface Expression
Studies examining TNF receptor expression on monocytes revealed that in the presence of TNF, both TNF-RI and TNF-RII surface levels were reduced by 2.4-fold. Notably, adalimumab at a concentration of 16.7 nM maintained TNF-RI at baseline levels when pre-formed with TNF, demonstrating receptor preservation. All anti-TNF biologics, including adalimumab, were equally effective in preventing the loss of surface TNF-RII.
Blocking antibody studies demonstrated that blocking TNF-RI curtailed TNF-dependent osteoclast precursor functions, indicating that TNF-RI is the receptor predominantly involved in TNF-enhanced osteoclast function and that adalimumab restricts TNF access to this receptor.
Functional Signaling Outcomes
Adalimumab binding to TNFα prevented the activation of TNFRs, which are involved in signaling pathways such as NF-κB. In U937 NF-κB luciferase reporter cells, TNF stimulation led to a 122-fold increase in luciferase activity, which adalimumab reduced to baseline levels at higher concentrations. The inhibition of proinflammatory signaling was demonstrated through blocking of TNF α-induced caspase activation and IL-8 secretion.
Adalimumab also completely inhibited TNF-induced apoptosis in a dose-dependent manner. TNF-enhanced ICAM-1 surface expression (3-fold increase) on U937 cells was reduced to baseline by 16.7 nM adalimumab. In primary CD14+ monocytes, adalimumab:TNF complexes not only prevented TNF induction of ICAM-1 but significantly reduced its level below baseline.
Studies also revealed that alpha-2,6 sialylation surface levels dropped in the presence of TNF specifically on the subset of cells undergoing apoptosis, and this subset was reduced proportionately to the inhibitory properties of adalimumab on apoptosis.
Fc-Mediated Functions
Adalimumab-TNF complexes activated FcγR-mediated signaling pathways, leading to increased clearance of antigens and antibodies and production of proinflammatory cytokines like interferon-γ. Reporter assays using Jurkat/FcγRIIa/NFAT-Luc and Jurkat/FcγRIIIa/NFAT-Luc cells showed that adalimumab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF. The assays involved mixing TNF antagonists with TNF at different molar ratios, incubating for 30 minutes at 37°C, then adding to Jurkat cells at a final antagonist concentration of 1 μg/mL, with luciferase activities measured using the ONE-Glo Luciferase Assay System.
Comparative Analysis with Other TNF Inhibitors
Binding Characteristics Comparison
Binding affinity measurements revealed differences among TNF inhibitors. Adalimumab demonstrated a binding affinity of 30.4 pM, compared to infliximab at 27.3 pM and etanercept at 11.8 pM. However, the epitope overlap with the TNFR2 binding site was more substantial for adalimumab than for infliximab. The larger buried surface area of adalimumab compared to infliximab and etanercept indicated a stronger interaction with TNFα, potentially leading to better efficacy in blocking receptor binding.
While etanercept exclusively formed 1:1 complexes with TNF, adalimumab formed larger, more complex assemblies. The mechanistic feature that distinguished adalimumab from etanercept was its ability as an antibody to form complexes with TNF, unlike the TNF receptor:Fc fusion protein etanercept. Interestingly, the Fc domain of adalimumab did not contribute to its suppressive function.
Functional Superiority
Head-to-head comparisons demonstrated that adalimumab and certolizumab pegol maintained baseline levels of TNF-RI more effectively than etanercept, infliximab, and golimumab. Adalimumab preserved 100% of TNF-RI, while etanercept preserved only 43%, infliximab 52%, and golimumab 62%. However, all agents were equally effective in preventing loss of surface TNF-RII, indicating no difference in selectivity for p75.
Adalimumab demonstrated superior efficacy across multiple functional outcomes. In NF-κB activation assays, adalimumab reduced luciferase activity to baseline, whereas etanercept achieved only partial reduction (3-fold), infliximab (7-fold), and golimumab (11-fold). Similarly, adalimumab reduced ICAM-1 surface expression to baseline, while etanercept was only 48% effective, infliximab 61%, and golimumab 59%.
For apoptosis inhibition, adalimumab and certolizumab completely inhibited TNF-induced apoptosis, unlike etanercept (42% reduction), infliximab (32% reduction), and golimumab (42% reduction). In FcγR activation, infliximab exhibited higher potency than adalimumab, with both showing greater activity than etanercept.
The clinical advantages of adalimumab over infliximab and etanercept were attributed to its direct inhibition mechanism through substantial overlap with the TNFα-TNFR2 interface, resulting in higher efficacy in autoimmune diseases.
Synthesis
The reviewed studies reveal a consistent mechanism whereby adalimumab blocks TNF receptor signaling primarily through competitive inhibition rather than direct receptor binding. No studies provided direct binding affinity measurements for adalimumab to isolated p55 or p75 TNF receptors. Instead, the evidence indicates that adalimumab exerts its effects by binding to TNFα and sterically blocking its interaction with cell surface receptors.
The differential preservation of TNF-RI versus TNF-RII by adalimumab compared to other agents can be mechanistically explained by the size and nature of immune complexes formed. Adalimumab forms larger complexes (3 antibodies:1-2 TNF trimers) compared to etanercept’s 1:1 complexes. These larger complexes may more effectively sequester TNF away from cell surfaces, preventing TNF-RI internalization that normally occurs upon TNF binding. The preservation is specific to TNF-RI, with all biologics equally protecting TNF-RII, suggesting that TNF-RI is more susceptible to TNF-induced downregulation under the conditions tested.
The superior functional outcomes of adalimumab compared to infliximab in monocyte assays appear paradoxical given that infliximab has slightly higher binding affinity (27.3 pM vs 30.4 pM). However, this is explained by epitope positioning: adalimumab’s epitope overlaps more substantially with the TNFα-TNFR2 binding interface, achieving more complete steric blockade despite marginally lower affinity. The larger buried surface area of 2,540 Ų may also contribute to more stable complex formation that resists dissociation under physiological conditions.
The finding that adalimumab’s Fc domain does not contribute to its inhibitory effects on osteoclast function appears inconsistent with the observed FcγR activation by adalimumab-TNF complexes. This apparent contradiction is resolved by recognizing context-dependent mechanisms: Fc-mediated functions like immune complex clearance and cytokine production occur at the systemic level, while direct inhibition of TNF-induced cellular responses (apoptosis, ICAM-1 expression, NF-κB activation) depends solely on blocking TNF access to its receptors through F(ab’)2-mediated binding. The F(ab’)2 fragment of adalimumab inhibited osteoclast activity to the same level as the whole IgG, confirming that receptor blockade is Fc-independent.
The consistent finding across multiple assay systems that adalimumab preferentially maintains TNF-RI expression while showing no selectivity for TNF-RII preservation suggests a receptor-specific mechanism. TNF-RI (p55) mediates most pro-inflammatory and apoptotic signals, making its preservation clinically relevant. The mechanistic basis likely involves TNF-RI’s more rapid internalization kinetics compared to TNF-RII; by forming stable complexes that prevent TNF from reaching the cell surface, adalimumab indirectly preserves receptor availability without directly binding these receptors.