IL33 Antibody

What is IL-33 and why is it an important research target?

IL-33 is a member of the IL-1 cytokine family that functions as an alarmin signal released during tissue damage or cellular necrosis. It is constitutively expressed in structural and lining cells including fibroblasts, endothelial, and epithelial cells of tissues exposed to the environment (skin, gastrointestinal tract, lungs) . IL-33 plays critical roles in both innate and adaptive immune responses, particularly in type 2 immunity and allergic airway diseases . It signals through the IL1RL1 (ST2) receptor and IL-1 receptor accessory protein (IL-1RAcP) complex, activating MyD88-dependent inflammatory pathways . Research interest in IL-33 has increased as genetic data demonstrates that individuals heterozygous for loss-of-function mutations have lower eosinophil counts and protection against asthma .

What are the basic structural and functional characteristics of IL-33?

IL-33 is encoded by the Il1rl1 gene and lacks a secretory signal peptide, preventing release through the classical endoplasmic reticulum and Golgi pathway . Key structural elements include:

• N-terminus containing a nuclear localization sequence

• Homeodomain-like helix-turn-helix DNA-binding domain

• Chromatin-binding domain

• C-terminal IL-1-like cytokine domain

Under homeostatic conditions, IL-33 (full-length, IL-33 FL) resides in cell nuclei associated with chromatin and functions as a transcriptional repressor . During cellular necrosis, tissue damage, or specific cellular activation, IL-33 is passively released in its full-length form (amino acids 1-270) . Functionally, IL-33 activates signaling pathways including nuclear factor kappa-B (NF-κB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) cascades in cells expressing the ST2 receptor .

What cell types express ST2 receptor and respond to IL-33?

ST2 receptor is selectively and stably expressed on multiple immune cell types that respond to IL-33 signaling :

What are the challenges in measuring IL-33 in biological samples?

Measuring IL-33 in biological samples presents several technical challenges :

• Interference from binding partners: Endogenous binding partners (e.g., soluble ST2) cause under-quantitation in commercial IL-33 kits.

• Redox state sensitivity: IL-33 exists in reduced (active) and oxidized forms with different biological activities and structural conformations.

• Low circulating levels: IL-33 is typically present at very low concentrations in serum/plasma.

• Rapid degradation: IL-33 is unstable and can be rapidly degraded by proteases in biological fluids.

To overcome these challenges, researchers should consider acid dissociation of samples to disrupt binding with endogenous partners, and simultaneous addition of detection reagent with the capture step. This modified approach enables detection of total reduced (active) IL-33 in human serum with a lower limit of quantitation (LLOQ) of 6.25 pg/ml .

How should researchers optimize IL-33 antibody-based detection assays?

Based on research findings, the following methodological considerations improve IL-33 detection :

• Acid treatment of samples: Acid dissociation of serum samples releases IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml.

• Simultaneous capture and detection: Adding detection reagent simultaneously with the capture step prevents re-association with binding partners.

• Specificity verification: Ensure the assay is specific for reduced (active) endogenous IL-33.

• Standardization: Use recombinant IL-33 standards that match the conformation state being measured.

• Sample handling: Minimize freeze-thaw cycles and process samples consistently to reduce variability.

What are the available antibody applications for IL-33 research?

Commercial anti-IL-33 antibodies support multiple research applications :

How do anti-IL-33 antibodies like tozorakimab function in neutralizing IL-33 activity?

Tozorakimab (MEDI3506) represents an advanced anti-IL-33 antibody with dual mechanisms of action :

• Direct neutralization of reduced IL-33 (IL-33ʳᵉᵈ):

  • Binds IL-33ʳᵉᵈ with femtomolar affinity (KD = 30 fM)

  • Fast association rate (8.5 × 10⁷ M⁻¹s⁻¹), comparable to soluble ST2

  • Prevents IL-33ʳᵉᵈ interaction with ST2 receptor

  • Inhibits ST2-dependent inflammatory responses

• Prevention of IL-33 oxidation:

  • Prevents conversion of IL-33ʳᵉᵈ to oxidized IL-33 (IL-33ᵒˣ)

  • Indirectly inhibits IL-33ᵒˣ signaling via RAGE/EGFR pathway

  • Increases epithelial cell migration and repair in vitro

These mechanisms allow tozorakimab to inhibit both ST2-dependent inflammation and RAGE/EGFR-mediated epithelial dysfunction .

What are critical antibody properties for effective IL-33 neutralization in vivo?

Research on tozorakimab development revealed specific antibody characteristics critical for effective IL-33 neutralization :

• High affinity binding: Antibody affinity should exceed that of soluble ST2 (sST2) for IL-33 (femtomolar range).

• Fast association rate: Association rates greater than 10⁷ M⁻¹s⁻¹ are required to effectively neutralize IL-33 following rapid release from damaged tissue.

• Epitope specificity: Antibodies should target epitopes that prevent IL-33 interaction with ST2 receptor.

• Conformational stability: Antibodies should maintain binding specificity under physiological conditions.

• Prevention of oxidation: Ability to prevent oxidation of IL-33 provides additional therapeutic benefit through dual mechanism of action.

In silico modeling demonstrated that while very high affinities between 0.1-10 pM had modest effects on free IL-33ʳᵉᵈ levels, association rates of 10⁷-10⁸ M⁻¹s⁻¹ were crucial for attenuating IL-33 spikes below 100% and 10% of steady-state levels, respectively .

How should researchers approach IL-33 antibody development and selection?

Based on successful development of tozorakimab, researchers should consider the following approach :

• Target stabilization: Use oxidation-resistant forms (e.g., IL-33 C>S with cysteine-to-serine substitutions) to preserve conformational epitopes during antibody generation.

• Diverse selection strategies: Employ multiple selection approaches including phage display with both wild-type and stabilized IL-33 forms.

• Competitive screening: Use competition assays with soluble ST2 to identify antibodies that block receptor interaction.

• Affinity maturation: Implement comprehensive affinity maturation through random mutagenesis of complementarity-determining regions (CDRs).

• Functional validation: Validate antibodies in cellular assays measuring both ST2-dependent signaling and downstream functional effects.

• In vivo modeling: Test neutralization capacity in models of acute tissue injury with rapid IL-33 release.

The development of tozorakimab required affinity optimization (>100,000-fold improvement) exploring sequence modifications across multiple CDRs .

What are the key signaling pathways activated by IL-33?

IL-33 activates multiple signaling pathways in different cell types, with distinct outcomes :

• ST2-dependent pathway:

  • Principal receptor for reduced IL-33 (IL-33ʳᵉᵈ)

  • Forms complex with IL-1 receptor accessory protein (IL-1RAcP)

  • Activates MyD88-dependent signaling

  • Triggers NF-κB, MAPK (p38, JNK, ERK) cascades

  • Induces type 2 cytokine production

• RAGE/EGFR pathway:

  • Activated by oxidized IL-33 (IL-33ᵒˣ)

  • Functions independently of ST2

  • Affects epithelial cell migration and repair

  • Contributes to tissue remodeling

• Nuclear functions:

  • Full-length IL-33 acts as transcriptional regulator

  • Associates with chromatin via N-terminal domain

  • Functions as transcriptional repressor

Understanding these distinct pathways is essential for designing research strategies targeting specific IL-33 functions .

How does IL-33 contribute to immune cell regulation in different tissues?

IL-33 regulates multiple immune cell types with tissue-specific effects :

• In lungs:

  • Activates ILC2s to produce IL-5 and IL-13

  • Promotes eosinophil recruitment and survival

  • Induces goblet cell hyperplasia and mucus production

  • Polarizes macrophages toward M2 phenotype

  • Contributes to airway hyperresponsiveness

• In skin:

  • Enhances production of pro-inflammatory cytokines (IL-6, CXCL8, CCL2)

  • Increases expression of adhesion molecules on eosinophils

  • Upregulates ICAM-1 on fibroblasts

  • Contributes to atopic dermatitis pathogenesis

• In lymphoid tissues:

  • Promotes B cell responses and antibody production

  • Induces BAFF (B cell activating factor) expression

  • Supports germinal center formation

  • Increases T follicular helper (TFH) cell numbers

• In synovium:

  • Contributes to rheumatoid arthritis inflammation

  • Correlates with autoantibody production

  • Levels decrease after successful anti-TNF treatment

The tissue-specific effects of IL-33 highlight the importance of context-dependent experimental design when studying IL-33 antibody interventions .

How does IL-33 influence autoimmunity and tolerance?

Research demonstrates complex roles for IL-33 in autoimmunity and tolerance :

• Breaking immune tolerance:

  • Short-term exposure to IL-33 can induce primary (IgM) responses to self-antigens

  • IL-33 drives BAFF production, promoting B cell survival

  • Chronic IL-33 exposure leads to class-switching from IgM to IgG autoantibodies

  • IL-33 increases T follicular helper (TFH) cell numbers and germinal center formation

• Cellular sources of BAFF:

  • Radiation-resistant cells (not myeloid cells) are major sources of IL-33-induced BAFF

  • BAFF neutralization prevents IL-33-induced increases in B cell numbers and autoantibody titers

• Disease associations:

  • Serum and synovial fluid IL-33 levels are elevated in rheumatoid arthritis patients

  • IL-33 levels correlate with production of rheumatoid factor and anti-citrullinated protein antibodies

  • Successful anti-TNF treatment reduces IL-33 levels in responsive patients

These findings indicate that IL-33 antibodies might have therapeutic potential in autoimmune conditions by interrupting the IL-33-BAFF axis .

What experimental models are appropriate for testing anti-IL-33 antibodies?

Based on the research literature, several experimental models are suitable for evaluating anti-IL-33 antibodies :

• Acute lung injury models:

  • Allergen challenge (Alternaria, papain, HDM)

  • Viral infection (influenza)

  • Chemical or mechanical injury

  • Effectively demonstrates rapid IL-33 release and alarmin activity

• Chronic airway inflammation models:

  • Multiple allergen exposures over 8-12 weeks

  • Models persistent IL-33 expression and tissue remodeling

  • Evaluates long-term antibody efficacy

• Autoimmunity models:

  • IL-33 injection protocols (4 daily doses)

  • AAV-vector driven chronic IL-33 expression

  • Measures autoantibody development and class-switching

• Epithelial repair models:

  • In vitro wound healing assays

  • Tests effects on RAGE/EGFR signaling pathway

  • Evaluates dual mechanisms of anti-IL-33 antibodies

When designing experiments, researchers should consider species-specific differences in IL-33 expression patterns. For example, mouse IL-33 is mainly expressed by alveolar type II pneumocytes, whereas human IL-33 is expressed by bronchial epithelial cells .

How do researchers evaluate the efficacy of anti-IL-33 antibodies in preclinical studies?

Comprehensive evaluation of anti-IL-33 antibodies requires multiple outcome measures :

• Biochemical parameters:

  • Binding affinity determination (KD)

  • Association/dissociation rate constants (kon, koff)

  • Epitope mapping and competition with sST2

  • Prevention of oxidation

• Cellular assays:

  • Inhibition of IL-33-induced NF-κB activation

  • Suppression of cytokine production (IL-5, IL-13, etc.)

  • Prevention of cellular signaling events

  • Effects on cell migration and tissue repair

• In vivo readouts:

  • Reduction in inflammatory cell infiltration

  • Suppression of inflammatory cytokines/chemokines

  • Prevention of tissue remodeling

  • Functional outcomes (e.g., airway hyperresponsiveness)

• Pharmacokinetic/pharmacodynamic analysis:

  • Antibody half-life determination

  • Target engagement assessment

  • Biomarker modulation (e.g., sST2 levels)

  • Dose-response relationships

Tozorakimab evaluation, for example, demonstrated femtomolar affinity (KD = 30 fM), complete inhibition of IL-33-dependent cellular activation, and effective neutralization in murine models of acute lung injury .

What are the key technical considerations for analyzing IL-33 expression in patient samples?

Research highlights several important technical considerations for analyzing IL-33 in clinical samples :

• Sample preparation:

  • Acid dissociation is required to detect IL-33 in the presence of binding partners

  • Serum/plasma samples need special handling to preserve IL-33 activity

  • Consider sampling route (BAL, serum, tissue biopsies) based on research question

• Assay specificity:

  • Distinguish between full-length and processed forms of IL-33

  • Differentiate between reduced (active) and oxidized IL-33

  • Account for potential interference from soluble ST2

• Tissue analysis:

  • IL-33 is primarily expressed in bronchial epithelium in humans

  • Nuclear localization is characteristic of intact cells

  • Extracellular IL-33 indicates tissue damage or active release

• Clinical correlation:

  • Compare IL-33 levels across different disease phenotypes

  • Consider disease activity and treatment status

  • Account for demographic factors (age, sex, ethnicity)

Interestingly, analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no significant difference in serum IL-33 levels , highlighting the importance of tissue-specific rather than systemic IL-33 measurement in respiratory diseases.

How do researchers distinguish between effects of different IL-33 isoforms?

IL-33 exists in multiple forms with distinct biological activities :

• Full-length IL-33 (IL-33 FL):

  • Nuclear localization and transcriptional regulation

  • Released during cell necrosis

  • Requires processing for optimal activity

• Reduced IL-33 (IL-33ʳᵉᵈ):

  • Contains reduced cysteine residues

  • Signals through ST2/IL-1RAcP complex

  • Potent inducer of type 2 immunity

• Oxidized IL-33 (IL-33ᵒˣ):

  • Contains disulfide bonds between cysteine residues

  • Signals through RAGE/EGFR complex

  • Affects epithelial cell function and repair

To distinguish between these forms, researchers should:

• Use conformation-specific antibodies

• Employ reducing vs. non-reducing conditions in assays

• Generate oxidation-resistant forms (e.g., IL-33 C>S) for comparison

• Evaluate both ST2-dependent and RAGE/EGFR-dependent readouts

• Consider dual-mechanism antibodies (like tozorakimab) that affect both pathways

The discovery that tozorakimab prevents oxidation of IL-33 and thereby inhibits IL-33ᵒˣ-dependent activities represents a significant advance in understanding IL-33 biology .

What are the current limitations in IL-33 antibody research?

Several challenges remain in IL-33 antibody research :

• Species differences:

  • Mouse IL-33 is mainly expressed in alveolar type II pneumocytes

  • Human IL-33 is primarily expressed in bronchial epithelial cells

  • These differences may affect translation of mouse models to humans

• Redox state complexity:

  • Oxidation significantly alters IL-33 structure and function

  • Most assays don't distinguish between reduced and oxidized forms

  • Environmental factors influence oxidation status

• Assay limitations:

  • Interference from binding partners in biological samples

  • Low circulating levels challenge detection sensitivity

  • Rapid degradation affects reproducibility

• Dual functions:

  • Nuclear vs. extracellular roles are difficult to separate experimentally

  • Cell-specific responses complicate interpretation

  • Beneficial vs. pathological effects depend on context

• Therapeutic targeting:

  • Complete IL-33 blockade may affect beneficial tissue repair functions

  • Optimal timing of intervention remains unclear

  • Patient stratification strategies need development

Researchers should consider these limitations when designing experiments and interpreting results in IL-33 antibody studies.

What are emerging research directions for IL-33 antibody development?

Based on current research, several promising directions are emerging :

• Conformation-specific antibodies:

  • Development of antibodies selective for specific IL-33 forms

  • Targeting distinct epitopes on reduced vs. oxidized IL-33

  • Pathway-selective inhibition strategies

• Dual-mechanism antibodies:

  • Further exploration of antibodies that both neutralize IL-33ʳᵉᵈ and prevent oxidation

  • Evaluation in chronic disease models

  • Assessment of tissue repair enhancement

• Tissue-targeted approaches:

  • Lung-specific delivery systems for respiratory diseases

  • Skin-targeted approaches for dermatological conditions

  • Tissue-selective expression of decoy receptors

• Biomarker development:

  • Identification of IL-33-responsive patient subgroups

  • Development of companion diagnostics

  • Correlation of tissue vs. systemic IL-33 expression

• Combination therapies:

  • IL-33 antibodies combined with other biologics

  • Sequential or alternating treatment strategies

  • Targeting multiple alarmins simultaneously

These emerging directions highlight the continuing evolution of IL-33 antibody research and its potential therapeutic applications.