IL17F Antibody

What is IL-17F and how does it differ from other IL-17 family cytokines?

IL-17F is a homodimeric, 34 kDa cytokine belonging to the IL-17 family, which includes IL-17A, IL-17B, IL-17C, IL-17D, and IL-17E (also known as IL-25). Among these family members, IL-17F shares the highest homology with IL-17A. Both IL-17A and IL-17F are predominantly produced by T helper 17 (Th17) cells, while other family members are produced more widely by different cell types .

Despite their structural similarities, IL-17F and IL-17A possess both overlapping and independent functions as demonstrated through knockout studies. IL-17F is involved in allergic airway inflammation and can induce several cytokines, chemokines, and adhesion molecules in bronchial epithelial cells, vein endothelial cells, fibroblasts, and eosinophils . The relative difference in potency between recombinant human IL-17A and IL-17F is approximately 100-fold, with IL-17A being more potent .

In inflammatory conditions, IL-17F may exist as a homodimer or as a heterodimer with IL-17A (IL-17A/F). Quantification studies in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis have revealed that IL-17F is significantly more abundant than IL-17A (>30-fold) .

How should researchers design experiments to validate IL-17F antibody specificity?

To validate IL-17F antibody specificity, researchers should implement a multi-step approach:

• Cross-reactivity testing: Evaluate binding to recombinant human IL-17 family members (IL-17A through IL-17E) using ELISA. Antibodies should demonstrate high specificity for IL-17F with minimal cross-reactivity to other family members, particularly IL-17A which shares the highest structural similarity .

• Competitive binding assays: Perform competitive binding experiments where unlabeled IL-17F or other IL-17 family cytokines compete with labeled IL-17F for antibody binding. A specific antibody will show displacement only with unlabeled IL-17F.

• Functional validation: Test the antibody's ability to neutralize IL-17F bioactivity in cell-based assays. For example, measure inhibition of IL-17F-induced IL-6 production in normal human dermal fibroblasts (NHDFs) stimulated with TNFα and IL-17F .

• Western blot and immunoprecipitation: Confirm specificity by detecting endogenous or recombinant IL-17F at the expected molecular weight (~34 kDa for homodimer).

• Knockout/knockdown controls: Include IL-17F knockout or knockdown samples as negative controls when possible to confirm specificity.

What are the recommended protocols for IL-17F detection in biological samples?

For optimal IL-17F detection in biological samples, researchers should consider these methodological approaches:

Flow cytometry (intracellular staining):

• Fix and permeabilize cells using a commercial kit designed for cytokine detection

• Use 5 μL (0.5 μg) of IL-17F antibody per test (10^5 to 10^8 cells) in 100 μL final volume

• Include protein transport inhibitors during cell stimulation (e.g., brefeldin A or monensin)

• For optimal results with PE-conjugated antibodies, use excitation at 488-561 nm and emission detection at 578 nm

ELISA detection:

• Coat high-binding ELISA plates with recombinant IL-17F or capture antibody

• For direct binding studies, add antibody titrations starting at 10 μg/mL

• Measure optical density at 450 nm

Cell stimulation protocols for IL-17F induction:

• For T cells: Stimulate with PMA/ionomycin for 4-6 hours with protein transport inhibitor

• For Th17 polarization: Culture naive CD4+ T cells with TGF-β, IL-6, IL-1β, IL-23, anti-IFN-γ, and anti-IL-4 for 5-6 days

Sample preparation considerations:

• Serum/plasma: Centrifuge blood samples within 30 minutes of collection

• Tissue samples: Homogenize in PBS with protease inhibitors before analysis

• Cell culture supernatants: Collect after appropriate stimulation period

How can researchers distinguish between IL-17F homodimers and IL-17A/F heterodimers in experimental systems?

Distinguishing between IL-17F homodimers and IL-17A/F heterodimers requires sophisticated experimental approaches:

Antibody-based discrimination:

• Use combination of specific antibodies targeting epitopes unique to IL-17F homodimers versus those present on heterodimers

• Implement sandwich ELISA systems with capture antibodies specific for one subunit and detection antibodies for the other subunit

Affinity purification:

• Perform sequential immunoprecipitation with anti-IL-17A and anti-IL-17F antibodies

• Analyze the resulting fractions by western blot to identify homodimers versus heterodimers

Functional characterization:

• Utilize differential receptor binding characteristics (IL-17RC binds IL-17F with higher affinity than IL-17A)

• Compare neutralization effects of selective antibodies against homodimers versus heterodimers in bioassays

Mass spectrometry:

• Implement proteomics approaches to identify unique peptide signatures from homodimers versus heterodimers

• Use crosslinking mass spectrometry to characterize dimeric interfaces

Research has shown that in vitro cultured Th17 cells can express IL-17F and IL-17A homodimers and IL-17A/F heterodimers depending on culture conditions and differentiation state . This heterogeneity must be considered when designing experimental systems targeting IL-17F biology.

What methodological considerations are important when comparing IL-17A versus IL-17F neutralization in disease models?

When designing experiments to compare IL-17A versus IL-17F neutralization in disease models, researchers should consider these methodological factors:

Antibody selection and validation:

• Ensure comparable affinity and neutralizing potency of anti-IL-17A and anti-IL-17F antibodies

• Validate neutralization capacity using in vitro bioassays before in vivo application

Dosing considerations:

• Account for differences in potency between IL-17A and IL-17F (~100-fold in humans)

• Implement dose-response studies to determine optimal antibody concentrations

• For combined neutralization studies, consider using 100 μg of each specific antibody daily via intraperitoneal administration (based on mouse studies)

Experimental design:

• Include appropriate control groups: isotype controls, single neutralization (anti-IL-17A or anti-IL-17F alone), and combination (anti-IL-17A + anti-IL-17F)

• Consider the temporal expression patterns: IL-17A may be induced earlier than IL-17F in disease progression

Readout selection:

• Measure both direct neutralization outcomes and downstream effects

• Monitor disease-specific parameters (e.g., arthritic score, airway inflammation)

• Assess changes in cytokine profiles, particularly IL-6 and G-CSF which are often suppressed following effective IL-17A neutralization

• Evaluate cellular infiltration patterns in affected tissues

Analysis of IL-17A/F ratios:

• Determine baseline ratios of IL-17F to IL-17A in your model system

• Consider that when the ratio of IL-17F to IL-17A is ≥10-fold, dual neutralization may show differentiated effects compared to IL-17A inhibition alone

How does affinity maturation impact IL-17F antibody performance in research applications?

Affinity maturation can dramatically enhance IL-17F antibody performance in research applications, as evidenced by the development of dual-specific antibodies like bimekizumab:

Effects on binding kinetics:
The affinity maturation process can significantly improve binding constants. For example, the bimekizumab development process demonstrated a 43-fold increase in affinity for IL-17F (from KD of 1510 pM to 35 pM) and a 4-fold increase in affinity for IL-17A (from KD of 29 pM to 7 pM) compared to its parent antibody .

Improvements in neutralization potency:
Affinity-matured antibodies demonstrate enhanced neutralization capacity in functional assays. In fibroblast stimulation assays with TNFα and IL-17F, the affinity-matured antibody 496.g3 showed significantly greater inhibition of IL-6 production compared to its parent antibody 496.g1 .

Impact on experimental sensitivity:
Higher affinity antibodies can:

• Detect lower concentrations of target protein

• Remain effective at lower dosages

• Provide more consistent results across experimental replicates

• Increase signal-to-noise ratio in detection applications

Methodological considerations:
When using affinity-matured antibodies, researchers should:

• Re-validate optimal working concentrations

• Consider potential off-target effects from increased binding to related proteins

• Adjust incubation times and washing protocols according to binding kinetics

• Document the specific clone and its affinity parameters in experimental reports

What are the key considerations for developing therapeutic antibodies targeting both IL-17A and IL-17F?

Developing therapeutic antibodies targeting both IL-17A and IL-17F presents several important technical and biological considerations:

Target biology understanding:

• Different tissue expression patterns of IL-17A and IL-17F

• Relative contributions of each cytokine to specific disease pathophysiology

• Potential redundancy or synergy between IL-17A and IL-17F functions

Antibody engineering approaches:

• Starting point selection: Researchers may begin with an IL-17A-specific antibody and enhance IL-17F binding through targeted mutations

• Affinity maturation strategies: For bimekizumab development, five mutations in the light chain variable region of the parent antibody enhanced binding to both cytokines

• Epitope selection: Target conserved epitopes between IL-17A and IL-17F while maintaining specificity within the IL-17 family

Pharmacokinetic/pharmacodynamic considerations:

• Higher antibody concentrations may be required for dual targeting compared to single-cytokine neutralization

• When the ratio of IL-17F to IL-17A is ≥10-fold (as observed in many inflammatory conditions), dual neutralization shows differentiated benefits

• Target-mediated drug disposition modeling helps predict tissue penetration and neutralization capacity

Functional validation strategies:

• Compare single versus dual neutralization in complex bioassays

• Test varying ratios of IL-17F:IL-17A to determine optimal neutralization conditions

• Evaluate inhibition across multiple cell types and readouts relevant to disease biology

The dual-targeting approach is supported by research showing that IL-17F is significantly more abundant than IL-17A in inflammatory conditions (>30-fold), and that when the ratio of IL-17F to IL-17A is ≥10-fold, dual neutralization demonstrates superior inflammatory suppression compared to IL-17A inhibition alone .

What factors influence IL-17F antibody performance in intracellular staining experiments?

Several critical factors can affect IL-17F antibody performance in intracellular staining experiments:

Cell stimulation protocols:

• Optimal stimulation conditions: PMA (50 ng/mL) + ionomycin (1 μg/mL) for 4-6 hours

• Protein transport inhibitor selection: Brefeldin A works better for most cytokines, including IL-17F

• Timing: IL-17F expression peaks later than some other cytokines, requiring longer stimulation

Fixation and permeabilization:

• Fixative selection: Paraformaldehyde (4%) preserves cellular morphology while maintaining epitope accessibility

• Permeabilization agent: Saponin-based buffers typically work well for cytokine detection

• Incubation times: Excessive permeabilization can reduce signal intensity

Antibody parameters:

• Optimal concentration: 5 μL (0.5 μg) per test for flow cytometric analysis

• Clone selection: The SHLR17 monoclonal antibody clone has been validated for intracellular staining of human IL-17F

• Fluorophore selection: PE conjugates (excitation: 488-561 nm; emission: 578 nm) provide good signal-to-noise ratio

Technical considerations:

• Cell number optimization: Test range from 10^5 to 10^8 cells per 100 μL test

• Buffer composition: Presence of serum proteins can reduce non-specific binding

• Washing steps: Insufficient washing can increase background

How can researchers optimize IL-17F neutralization experiments to study inflammatory pathways?

To optimize IL-17F neutralization experiments for studying inflammatory pathways:

Cell model selection:

• Normal human dermal fibroblasts (NHDFs) respond robustly to IL-17F stimulation, especially in combination with TNFα

• Bronchial epithelial cells are appropriate for studying airway inflammation models

• Primary cells generally provide more physiologically relevant responses than cell lines

Stimulation conditions:

• Use IL-17F in combination with TNFα for synergistic induction of inflammatory mediators

• For human cell models: IL-17F (25 nM) combined with TNFα (0.025 nM) effectively stimulates IL-6 production

• Pre-incubate neutralizing antibodies with cytokines for 1 hour before addition to cells

• Standard stimulation period: 18-20 hours for robust cytokine production

Readout selection:

• Primary readouts: IL-6 production serves as a reliable indicator of IL-17F activity

• Additional informative markers: CXCL1, CXCL8, and CCL20 production

• Detection method: Homogeneous time-resolved fluorescence provides sensitive quantification

Experimental controls:

• Include IL-17F alone, TNFα alone, and combination conditions to demonstrate synergy

• Include isotype control antibodies at equivalent concentrations

• For dual-neutralization studies, include single-neutralization conditions (anti-IL-17A or anti-IL-17F alone)

Antibody titration:

• Perform dose-response experiments with neutralizing antibodies

• For combined IL-17A/F neutralization, test different antibody ratios and concentrations

What are the optimal strategies for evaluating IL-17F antibody efficacy in animal models of inflammation?

For evaluating IL-17F antibody efficacy in animal disease models, researchers should implement these strategic approaches:

Model selection considerations:

• Collagen-induced arthritis (CIA) provides a well-characterized model for studying IL-17 biology

• House dust mite (HDM) and toluene diisocyanate (TDI) models are effective for studying airway inflammation

• Consider species differences: The potency difference between mouse IL-17A and IL-17F is ~10,000-fold, compared to ~100-fold in humans

Administration protocol:

• Dosage: 100 μg of specific antibody daily via intraperitoneal route

• Control groups: Include isotype control antibodies (100 μg) for each specific antibody

• For evaluating combined effects: Administer 100 μg each of anti-IL-17A and anti-IL-17F antibodies

• Timing: In CIA models, initiate treatment from day 20 post-immunization

Assessment parameters:

• Clinical scoring: Monitor disease-specific parameters (e.g., arthritic score)

• Cytokine profiling: Measure serum levels of IL-6, G-CSF, IFN-γ, IL-1β, TNF-α, and GM-CSF

• Cellular analysis: Evaluate inflammatory cell infiltration by flow cytometry

• Histopathological evaluation: Assess tissue damage and inflammation

Temporal considerations:

• Monitor the differential induction timing of IL-17A and IL-17F

• In CIA models, IL-17A is induced first (during initiation phase), while IL-17F appears with the onset of arthritis

• Only a small percentage of CD4+ T cells co-express both IL-17A and IL-17F

Data interpretation:

• Compare single versus combined neutralization effects

• Evaluate systemic versus local effects of neutralization

• Consider unexpected findings: Some studies report increased IL-6 levels with anti-IL-17F antibody treatment alone

How should researchers interpret contradictory findings between IL-17F neutralization studies?

When faced with contradictory findings in IL-17F neutralization studies, researchers should systematically evaluate these factors:

Species and model differences:

• Mouse versus human IL-17F potency varies dramatically (~10,000-fold difference in mice versus ~100-fold in humans compared to IL-17A)

• Disease model selection affects cytokine dominance patterns

• Genetic background of animal models influences IL-17 pathway activities

Antibody characteristics:

• Affinity differences between antibodies used in different studies

• Epitope specificity affecting neutralization of different forms (monomer, homodimer, heterodimer)

• Isotype differences impacting in vivo half-life and effector functions

Experimental timing:

• IL-17A is induced earlier than IL-17F in some inflammatory models

• Intervention timing relative to disease phase (preventive versus therapeutic)

• Duration of neutralization treatment

Readout selection:

• Direct versus indirect measures of IL-17F activity

• Cellular versus molecular endpoints

• Acute versus chronic outcome measures

Contextual cytokine environment:

• Ratio of IL-17F to IL-17A in the specific model (~30-fold higher IL-17F in human inflammatory conditions)

• Presence of synergizing factors like TNFα

• Compensatory mechanisms in chronic neutralization settings

For example, in collagen-induced arthritis studies, anti-IL-17F antibody treatment alone did not reduce arthritis severity, while anti-IL-17A or combined anti-IL-17A/F antibody treatment significantly reduced disease. Surprisingly, IL-6 levels were actually increased in mice receiving anti-IL-17F antibody compared to isotype controls, suggesting complex regulatory mechanisms .

What quantitative approaches best measure IL-17F antibody efficacy in complex biological systems?

Quantitative assessment of IL-17F antibody efficacy in complex biological systems requires multi-dimensional analysis approaches:

Pharmacokinetic/pharmacodynamic modeling:

• Target-mediated drug disposition models predict antibody distribution and IL-17F occupancy in different tissues

• Allometric scaling to predict human pharmacokinetic parameters from animal data

• Simulation of percentage binding at different doses and intervals

Dose-response relationship analysis:

• EC50 determination for neutralization of IL-17F-induced cellular responses

• Comparison of potency shifts in the presence of other cytokines (e.g., TNFα)

• Neutralization efficacy at different IL-17F:IL-17A ratios

Multiparameter biomarker assessment:

• Cytokine network analysis: Measure changes in multiple downstream cytokines (IL-6, G-CSF)

• Transcriptomics: Evaluate global gene expression changes following neutralization

• Phosphoprotein analysis: Quantify changes in IL-17 signaling pathway activation

Mathematical modeling of pathway inhibition:

• Systems biology approaches to model IL-17 pathway dynamics

• Network analysis to identify key nodes affected by IL-17F neutralization

• Predictive modeling of therapeutic outcomes based on baseline parameters

Standardized reporting metrics:

• Neutralization potency (IC50 values)

• Receptor occupancy percentages in different tissues

• Biomarker modulation indices (percent change from baseline)

• Area under the effect curve (AUEC) for temporal integration of responses

For example, when developing bimekizumab, researchers used target-mediated drug disposition modeling to predict that following 160 mg IV dosing every 4 weeks, IL-17A would be completely bound in plasma and >95% bound in skin, but IL-17F would show <50% occupancy in skin. This informed subsequent affinity maturation efforts to improve IL-17F binding .

What are emerging techniques for studying IL-17F biology with enhanced precision?

Several cutting-edge approaches are enhancing precision in IL-17F research:

Single-cell analysis technologies:

• Single-cell RNA sequencing to identify discrete IL-17F-producing cell populations

• Mass cytometry (CyTOF) for high-dimensional characterization of IL-17F-producing cells

• Imaging mass cytometry to visualize IL-17F production in tissue microenvironments

Advanced protein engineering:

• Bispecific antibody formats targeting IL-17F and synergistic partners

• Conditional activation systems for temporal control of IL-17F neutralization

• Tissue-targeted antibody delivery to improve local neutralization efficiency

In vivo imaging techniques:

• Immuno-PET with radiolabeled anti-IL-17F antibodies to track tissue distribution

• Intravital microscopy to visualize IL-17F neutralization dynamics in real-time

• Bioluminescence reporters to monitor IL-17F pathway activation longitudinally

Computational approaches:

• Machine learning algorithms to predict IL-17F-dependent gene networks

• Molecular dynamics simulations of antibody-cytokine interactions

• Systems pharmacology models integrating multi-omics data

CRISPR-based methodologies:

• Precise genetic manipulation of IL-17F and receptor components

• CRISPR activation/repression systems for temporal control of IL-17F expression

• Base editing to introduce specific mutations in IL-17F pathway components

These advanced technologies will help address remaining questions about IL-17F biology, including: tissue-specific roles, temporal dynamics of expression, heterodimer versus homodimer functional differences, and optimal targeting strategies for different inflammatory conditions.

How might understanding of IL-17F biology inform development of next-generation therapeutic antibodies?

Deepening understanding of IL-17F biology is driving several key areas for next-generation therapeutic antibody development:

Pathway-selective targeting:

• Designing antibodies that selectively inhibit specific downstream pathways activated by IL-17F

• Developing antibodies that preferentially neutralize either homodimers or heterodimers

• Creating antibodies with differential tissue distribution profiles

Combination targeting strategies:

• Dual-variable domain antibodies targeting IL-17F and synergistic cytokines

• Bispecific antibodies neutralizing IL-17F while simultaneously engaging immune modulatory receptors

• Antibody-cytokine fusion proteins combining IL-17F neutralization with delivery of anti-inflammatory cytokines

Enhanced tissue targeting:

• Antibody engineering for improved blood-brain barrier penetration in neuroinflammatory conditions

• Pulmonary delivery systems for targeting airway inflammation

• pH-responsive antibodies for enhanced function in inflammatory microenvironments

Potency and selectivity optimization:

• Structure-guided design targeting unique epitopes on IL-17F

• Affinity maturation strategies focusing on tissue-specific efficacy

• Engineering for improved binding kinetics rather than equilibrium binding constants

Predictive biomarkers:

• Developing companion diagnostics to identify patients likely to benefit from IL-17F targeting

• Establishing IL-17F:IL-17A ratio thresholds predictive of dual-targeting efficacy

• Identifying genetic polymorphisms affecting IL-17F pathway activation

The understanding that IL-17F is significantly more abundant than IL-17A in inflammatory conditions (>30-fold) , and that dual neutralization shows superior effects at IL-17F:IL-17A ratios ≥10:1, suggests that antibodies effectively targeting both cytokines may provide enhanced therapeutic benefits for conditions like psoriasis, psoriatic arthritis, and ankylosing spondylitis.