IL25 Antibody

What is IL-25 and how does it function in immune pathways?

IL-25 is a member of the IL-17 family of cytokines that functions as a potent activator of type-2 immune responses. It induces the expression of several key inflammatory cytokines including IL-4, IL-5, and IL-13 . IL-25 mediates its biological effects through a receptor complex consisting of IL-17BR (IL-17RB) and IL-17RA, both of which are essential for IL-25 effector functions .

Multiple studies using knockout mice have demonstrated that mice deficient in either IL-17RA (Il17ra−/−) or IL-17RB (Il17rb−/−) fail to produce relevant inflammatory factors upon IL-25 stimulation. Following intranasal IL-25 administration, these knockout mice show no changes in inflammatory cells (eosinophils, neutrophils, lymphocytes, and macrophages) and no pathological alterations in lung tissue . This confirms that both receptor components are required for IL-25 signaling activation.

IL-25 is expressed in various tissues including lung, stomach, small intestine, and colon, and is produced by cell types such as macrophages, epithelial cells in the gut, Th2 cells, mast cells, and NKT cells .

What experimental methods are used to assess IL-25 antibody specificity?

Assessing IL-25 antibody specificity involves multiple complementary approaches:

• ELISA-based cross-reactivity testing: Plates are coated with potential cross-reactive proteins including IL-17BR-Fc, IL-17RA, IL-17RC-Fc, IL-17RD (SEF), or control proteins like IL-13Rα-Fc to test antibody binding specificity .

• Functional neutralization assays: Researchers evaluate the ability of antibodies to neutralize IL-25-induced effects in cell culture systems, measuring downstream cytokine production (IL-4, IL-5, IL-13) .

• Comparative binding studies: Binding affinity and specificity can be assessed against both human and mouse orthologs of IL-25 to determine species cross-reactivity, which is particularly important for translational research .

When developing the 22C7 antibody, researchers specifically engineered it to have equivalent in vitro affinity and potency against both human and mouse IL-25 orthologs, enabling consistent experimental results across species-based models .

How can researchers effectively evaluate IL-25 antibody efficacy in disease models?

Evaluating IL-25 antibody efficacy requires comprehensive assessment across multiple parameters:

Researchers should incorporate multiple disease models to comprehensively evaluate antibody efficacy. For example, studies have demonstrated that anti-IL-25 antibodies show efficacy in:

• Oxazolone-induced colitis models (mimicking ulcerative colitis)

• IL-25-induced air pouch models (assessing inflammatory cell recruitment)

• Aldara-induced psoriasiform skin inflammation models

• Allergic asthma models with eosinophilic inflammation

Combining these models provides more robust evidence of therapeutic potential across different inflammatory conditions .

What are the molecular mechanisms through which IL-25 neutralizing antibodies affect downstream signaling pathways?

IL-25 neutralizing antibodies disrupt multiple downstream signaling pathways that contribute to inflammatory disease pathology:

• TRAF6-NF-κB Pathway: IL-25 typically activates TRAF6, leading to NF-κB pathway activation. Neutralizing antibodies significantly diminish this activation, reducing pro-inflammatory gene expression .

• Co-stimulatory Molecule Expression: Recent research demonstrates that IL-25 upregulates important co-stimulatory molecules on antigen-presenting cells, particularly eosinophils. These include HLA-DR, PD-L1, and OX-40L . Anti-IL-25 antibodies prevent this upregulation, thereby reducing T cell activation.

• Type-2 Cytokine Production: IL-25 neutralization blocks the recruitment and activation of type-2 cytokine-producing cells, including eosinophils and Th2 cells, preventing amplification of allergic and inflammatory responses .

• Antigen Processing Modulation: IL-25 enhances antigen uptake by eosinophils, promoting their antigen-presenting capacity. Anti-IL-25 antibodies reduce this uptake, as demonstrated by studies using fluorescent DQ-OVA, where IL-25-deficient mice displayed significantly reduced antigen processing by eosinophils .

These mechanisms collectively explain why IL-25 neutralization provides therapeutic benefit across multiple inflammatory disease models characterized by type-2 immune dysregulation.

How do genetic knockout models compare with antibody neutralization approaches in IL-25 research?

The comparison between genetic knockout models and antibody neutralization approaches reveals important methodological considerations:

Studies using IL-25-/- mice have revealed that IL-25 deficiency significantly reduces:

• Eosinophil recruitment to lungs after allergen challenge

• Antigen uptake and processing by pulmonary eosinophils (demonstrated by reduced DQ-OVA+ eosinophils and lower mean fluorescence intensity)

• Allergic pulmonary inflammation in response to house dust mite allergens

Complementary antibody neutralization studies with clones like 2C3 have shown improved pathology in mouse models of colitis and asthma , while the 22C7 antibody demonstrated efficacy in skin inflammation models . The convergence of results from both approaches strengthens evidence for IL-25's critical role in these inflammatory conditions.

What methodological considerations are important when using IL-25 antibodies for in vivo studies?

When designing in vivo studies with IL-25 antibodies, researchers should consider several critical methodological factors:

• Antibody Dose Optimization: Studies have used varying doses depending on the model and route of administration. For intranasal administration, studies have used 1 μg/mouse of recombinant IL-25 to induce inflammation , while antibody doses must be optimized to effectively neutralize this amount.

• Timing of Administration:

  • Preventive protocols: Antibody administration before disease induction

  • Therapeutic protocols: Administration after disease establishment

  For example, in colitis models, antibodies are typically administered before and during oxazolone challenge to prevent disease onset .

• Assessment Timepoints: Different parameters peak at different timepoints following challenge. Studies have shown that eosinophil infiltration after HDM sensitization peaks at specific timepoints that must be carefully determined for each model .

• Appropriate Controls: Studies should include:

  • Isotype control antibodies to control for non-specific antibody effects

  • Comparative controls with established treatments where appropriate

  • Both wild-type and IL-25-deficient animals when possible for comprehensive analysis

• Combined Readouts: To thoroughly assess efficacy, researchers should examine:

  • Histopathological changes (tissue damage, cell infiltration)

  • Inflammatory cell profiles using flow cytometry

  • Cytokine production in tissue culture supernatants

  • Clinical parameters (weight loss, ear thickness, airway reactivity)

How can researchers dissect the contribution of IL-25 signaling in complex inflammatory diseases with mixed immune signatures?

Dissecting IL-25's specific contribution in complex inflammatory diseases requires sophisticated experimental approaches:

• Cell-specific conditional knockout models: Generate mice with cell-specific deletion of IL-25 or its receptors (IL-17RA/IL-17RB) to identify which cellular sources or responders are critical in different disease contexts.

• Temporal neutralization experiments: Administer anti-IL-25 antibodies at different disease stages to determine when IL-25 signaling is most critical - during initiation, propagation, or resolution phases.

• Co-neutralization approaches: Combine IL-25 neutralization with blockade of other inflammatory pathways (e.g., IL-17A, IL-13) to assess synergistic effects or compensatory mechanisms.

• Adoptive transfer studies: Transfer specific cell populations from wild-type or IL-25-deficient mice to appropriate recipients to identify which IL-25-responsive cells mediate pathology.

• Transcriptomic and proteomic profiling: Compare gene and protein expression profiles in tissues from control, diseased, and anti-IL-25-treated animals to identify specific pathways affected by IL-25 neutralization.

This approach has yielded important insights about IL-25's unique role. For example, unlike IL-17A which is critical for antimicrobial peptide production and host defense against pathogens like Staphylococcus aureus in the skin, IL-25 does not participate in antimicrobial responses, as demonstrated by studies showing no impaired pathogen resistance in IL-25-deficient mice . This illustrates how careful experimental design can distinguish between the functions of related cytokines.

What are the technical challenges in generating and validating humanized anti-IL-25 antibodies for translational research?

Developing humanized anti-IL-25 antibodies for translational research presents several technical challenges:

• Cross-species reactivity engineering: Creating antibodies with dual reactivity against human and mouse IL-25 is technically challenging but essential for translational studies. The 22C7 antibody exemplifies successful engineering of equivalent affinity and potency against both orthologs .

• Maintaining affinity through humanization: When engineering a fully human antibody from a parent molecule, maintaining the original binding affinity and potency is difficult. This requires sophisticated antibody engineering techniques and extensive validation .

• Functional assessment beyond binding: Researchers must evaluate not just binding affinity but functional neutralization capacity. This requires developing appropriate bioassays that reflect the complex biology of IL-25 signaling .

• Specificity validation: Anti-IL-25 antibodies must be rigorously tested for cross-reactivity with other IL-17 family members. This typically involves testing binding to recombinant proteins including IL-17RA, IL-17RC-Fc, IL-17RD, and control proteins .

• Stability and manufacturability assessment: Development of a "highly developable" antibody requires evaluation of thermal stability, aggregation propensity, expression levels, and other pharmaceutical properties beyond simple efficacy testing .

The successful development of 22C7, which maintained efficacy through humanization while preserving affinity and potency of the parent molecule, demonstrates that these challenges can be overcome with appropriate antibody engineering approaches .