Recombinant Human Interleukin-1 receptor accessory protein (IL1RAP), partial

What is the basic structure of human IL1RAP?

IL1RAP is a member of the Interleukin-1 receptor family. Mature human IL1RAP consists of a 347 amino acid extracellular domain (ECD) with three Ig-like domains, a 21 amino acid transmembrane segment, and a 182 amino acid cytoplasmic domain . The recombinant form typically consists of the extracellular domain spanning from Ser21 to Glu359 . IL1RAP exists in multiple isoforms, including membrane-bound (mIL1RAP) and soluble (sIL1RAP) forms, with the latter being formed by the extracellular domain of mIL1RAP .

What are the key functional domains of IL1RAP and how do they contribute to its signaling capabilities?

The functional architecture of IL1RAP contains several critical domains that determine its signaling capabilities:

• Extracellular Region: Contains a small hydrophobic patch formed by three amino acid residues: Ile 135, Leu 180, and Ile 181, which is essential for binding to IL-1Rs .

• D2 and D3 Domains: These extracellular domains interact with corresponding D2 and D3 domains of primary receptors . While initially characterized for IL-36R interactions, similar binding patterns likely occur with other IL-1 family receptors due to conserved cysteine residues that form disulfide bonds promoting the 3D structure of receptors .

• Transmembrane Domain: Anchors the protein in the cell membrane.

• Cytoplasmic Domain: Critical for downstream signaling through recruitment of adaptor proteins like MyD88 and IRAK-4, leading to MAPK pathway activation .

The functional importance of these domains is evident in experiments where disruption of IL1RAP interactions inhibits signaling through multiple pathways, including IL-1, IL-33, and IL-36 signaling cascades.

Where is IL1RAP normally expressed in healthy human tissues?

IL1RAP is ubiquitously expressed across multiple tissues including the liver, placenta, and white blood cells . In the hematopoietic system, IL1RAP is expressed at lower levels on normal hematopoietic stem and progenitor cells (HSPCs) compared to their malignant counterparts . The soluble form (sIL1RAP) is found in circulation at relatively high concentrations, with a reported median of approximately 300 ng/mL (range: 92–600 ng/mL) in healthy individuals . Expression varies across different immune cell types, with differential expression observed on monocytes, neutrophils, T cells, and macrophages as demonstrated by flow cytometry .

How is IL1RAP expression regulated in normal versus malignant cells?

IL1RAP is significantly upregulated in various hematological malignancies, particularly in leukemic stem cells, compared to normal counterparts. Expression analysis across different leukemia subtypes reveals distinct patterns:

Gene expression correlation analyses show that high IL1RAP expression in AML is associated with "mitochondrial translation elongation and termination," "energy production via oxidative phosphorylation," and a "leukemic granulocyte-monocyte progenitor signature," whereas low IL1RAP expression correlates with "regulation of RNA metabolic processes," "gene expression," and a glycolysis-enriched HSC-like signature .

What methodologies are most effective for measuring IL1RAP expression?

Multiple complementary techniques provide comprehensive analysis of IL1RAP expression:

• Flow Cytometry: Quantifies surface protein expression using fluorescently-labeled antibodies, measuring mean fluorescence intensity (MFI). This approach can detect co-expression with other surface markers (CD135, CD123, CD117) .

• RNA Sequencing: Measures transcript levels and enables correlation with genome-wide expression patterns. Methods include batch correction, normalization via LogNormalize method, and scaling by a factor of 10,000 followed by log1p-transformation .

• Quantitative Proteomics: Assesses protein abundance, with the advantage of detecting post-translational modifications.

• Gene-Based Clustering: Employs modularity optimization methods using graph-based approaches to identify gene expression patterns throughout disease progression .

• Western Blotting: Evaluates protein expression in cell lysates, particularly useful for detecting different IL1RAP isoforms.

For comprehensive analysis, researchers should combine multiple methods to verify consistency across transcriptional and protein levels.

What are standard methods for producing recombinant human IL1RAP protein?

Production of high-quality recombinant human IL1RAP involves several critical steps:

• Expression Construct Design: Most commonly involves cloning the extracellular domain (ECD, Ser21-Glu359) with a C-terminal 6-His tag for purification .

• Expression System Selection: Mammalian expression systems (e.g., ExpiCHO cells) are preferred to ensure proper folding and post-translational modifications .

• Purification Strategy: Affinity chromatography using the His-tag, followed by additional purification steps to achieve >90% purity.

• Quality Control: Size exclusion chromatography (SEC) and SDS-PAGE to verify purity and molecular weight .

• Formulation and Storage: Typically lyophilized from a 0.2 μm filtered solution in PBS and reconstituted at 200 μg/mL in PBS .

For carrier-free preparations (without BSA), special consideration must be given to protein stability during storage and handling .

What functional assays are recommended to evaluate recombinant IL1RAP activity?

Several complementary assays provide robust assessment of IL1RAP functionality:

• Binding Assays:

  • Radiolabeled ligand binding ([125I]-hrIL-1beta) to cells co-expressing IL1RAP and primary receptors, measuring saturable high-affinity binding .

  • Competitive binding with antibodies to IL1RAP (e.g., mAb 4C5) or primary receptors to confirm specificity .

• Signaling Assays:

  • Phospho-flow cytometric analysis using NFκB phosphorylation as a marker for IL1RAP-mediated signaling upon IL-1 or IL-33 stimulation .

  • Verification of specificity using IL-1 receptor antagonist (IL1RA) or blocking antibodies (e.g., mAb3F8) .

• Functional Cellular Assays:

  • Colony-forming cell (CFC) assays to evaluate impact on progenitor cell function .

  • Cell proliferation assays comparing growth with and without IL1RAP modulation .

  • Growth curves after serum depletion with and without IL-1β stimulation .

• Co-immunoprecipitation Studies:

  • Evaluation of physical interactions between IL1RAP and other receptors (IL-1R1, FLT3, c-KIT) .

Each assay should include appropriate positive and negative controls to ensure specificity and reproducibility of results.

How is IL1RAP implicated in hematological malignancies?

IL1RAP plays multifaceted roles in hematological malignancies beyond its canonical function in IL-1 signaling:

• Co-receptor Function Expansion: IL1RAP physically interacts with and mediates signaling through FLT3 and c-KIT, two receptor tyrosine kinases with established roles in AML pathogenesis .

• Leukemic Stem Cell Support: IL1RAP is consistently overexpressed on leukemic stem cells across various genetic subtypes of AML, CML, and high-risk MDS .

• IL-1 Pathway Activation: In CML, primitive (CD34+CD38−) cells express functional IL-1 receptor complexes and respond with NF-κB activation and marked proliferation in response to IL-1, effects that can be blocked by IL1RAP antibodies .

• Growth Advantage Mechanism: Knockdown of IL1RAP significantly reduces colony-forming capacity of primary MDS/AML cells, indicating its role in promoting leukemic cell growth and survival .

Flow cytometry analysis of 124 primary AML patients revealed that IL1RAP is frequently co-expressed with other important signaling receptors:

• 21% of patients were CD135+IL1RAP+

• 4% expressed IL1RAP without CD135

• 29.8% were IL1RAP+CD117+

• 28.1% were IL1RAP+CD123+

These co-expression patterns suggest potential synergistic effects between IL1RAP and other oncogenic signaling pathways.

What is the role of IL1RAP in inflammatory and metabolic diseases?

Beyond hematological malignancies, IL1RAP is implicated in several inflammatory and metabolic conditions:

• Atherosclerosis: IL1RAP blockade limits plaque development and inflammation in apolipoprotein E–deficient (Apoe−/−) mice, suggesting its role in atherosclerotic disease progression .

• Myocarditis: Studies in mouse models of coxsackievirus B3 (CVB3)–mediated and experimental autoimmune myocarditis (EAM) showed that IL1RAP blockade reduced disease severity and preserved cardiac function .

• Non-alcoholic Fatty Liver Disease (NAFLD): The QSOX1/IL1RAP ratio has been identified as a novel biomarker for NAFLD severity, with elevated QSOX1 and reduced IL1RAP levels associated with increasing disease severity .

• Systemic Sclerosis: IL1RAP has been implicated in fibrotic processes, with therapeutic antibodies targeting IL1RAP under development for treatment of inflammatory and fibrotic diseases .

In these conditions, IL1RAP appears to function as an amplifier of inflammatory signaling by mediating the effects of multiple IL-1 family cytokines simultaneously.

What targeting strategies have been developed against IL1RAP?

Several approaches for targeting IL1RAP have been developed and evaluated in preclinical and clinical settings:

• Blocking Antibodies:

  • Non-depleting IL1RAP blocking antibodies (e.g., CAN10) that inhibit IL-1, IL-33, and IL-36 signaling pathways simultaneously .

  • Antibodies that specifically block IL-1-induced effects without immune cell depletion .

• ADCC-Mediating Antibodies:

  • Antibodies designed to induce antibody-dependent cellular cytotoxicity against IL1RAP-expressing cells .

• Genetic Approaches:

  • shRNA-mediated knockdown of IL1RAP expression .

  • CRISPR/Cas9-mediated genetic deletion .

• T Cell Engagers:

  • IL1RAP-specific T cell engagers to deplete leukemic stem cells .

These approaches have shown efficacy in various preclinical models, with several advancing to clinical development. The first-in-human phase 1 dose-escalation study of CAN04 (an IL1RAP-targeting antibody) demonstrated that it can be safely administered to patients up to 10.0 mg/kg weekly .

What methodologies are used to evaluate the efficacy of IL1RAP-targeting therapies?

Comprehensive evaluation of IL1RAP-targeting therapies requires multi-parameter assessment:

• In Vitro Assays:

  • Colony formation assays measuring progenitor cell function .

  • Cell proliferation and differentiation assessments .

  • Pathway-specific signaling readouts (e.g., phospho-flow cytometry for NFκB activation) .

• In Vivo Disease Models:

  • Leukemia models: Patient-derived xenografts in immunodeficient mice .

  • Inflammatory models: Viral-induced or autoimmune myocarditis models, atherosclerosis models in Apoe−/− mice .

• Efficacy Parameters:

  • Disease-specific endpoints: Leukemic burden, plaque development, cardiac function.

  • Molecular endpoints: Changes in signaling pathway activation, gene expression profiles.

  • Cellular endpoints: Immune cell infiltration, differentiation status.

• Advanced Analytical Methods:

  • Spatial transcriptomics to analyze gene expression landscapes in treated versus control tissues .

  • Multi-parameter flow cytometry to assess effects on specific cell populations .

  • Echocardiography for functional assessment in cardiovascular models .

For myocarditis models, IL1RAP blockade demonstrated efficacy without impeding viral clearance from the heart while significantly reducing the numbers of infiltrating immune cells (CD4+ T cells, Ly6C+CCR2+ monocytes, neutrophils, and eosinophils) . In atherosclerosis models, treatment with an IL1RAP-blocking antibody limited plaque development and inflammation .

How can researchers optimize the stability and activity of recombinant IL1RAP?

Optimal handling of recombinant IL1RAP requires attention to several factors:

• Storage Conditions:

  • For lyophilized protein: Store at -20°C to -80°C in a manual defrost freezer.

  • Avoid repeated freeze-thaw cycles to maintain protein integrity .

• Reconstitution Protocol:

  • Reconstitute at the recommended concentration (200 μg/mL) in appropriate buffer (PBS) to ensure proper folding .

  • Allow complete reconstitution before aliquoting to maintain consistency.

• Carrier Protein Considerations:

  • BSA is commonly added as a carrier protein to enhance stability and shelf-life.

  • For applications where BSA might interfere, carrier-free preparations should be used .

• Quality Control Testing:

  • Functional testing using binding assays to confirm activity before use in experiments.

  • Assessment of aggregation state by SEC or dynamic light scattering.

• Experimental Design Factors:

  • When using recombinant IL1RAP as a reagent, consider its concentration-dependent effects.

  • For inhibition studies, the effective concentration range is 0.8-4 µg/mL in the presence of 1 μg/mL of recombinant human IL-1R2 Fc Chimera and 50 pg/mL rhIL-1 alpha .

What are the critical considerations for developing IL1RAP-targeting antibodies for research applications?

Development of effective IL1RAP-targeting antibodies requires careful consideration of several factors:

• Epitope Selection:

  • Target functional domains involved in receptor interactions to effectively block signaling.

  • The small hydrophobic patch (Ile 135, Leu 180, and Ile 181) and D2/D3 domains are critical for receptor interactions .

• Antibody Generation Methods:

  • Immunization with recombinant extracellular domain (ECD, S21-E359) of human IL1RAP .

  • Screening of plasma B cells from immunized mice using optofluidic systems (e.g., Beacon®) .

  • Recovery and sequencing of variable heavy (VH) and light (VL) chain sequences .

• Engineering Considerations:

  • Creation of mouse-human chimeric or fully humanized antibodies for therapeutic applications.

  • Fc modifications to enhance or eliminate effector functions depending on the desired mechanism of action .

• Functional Characterization:

  • Confirmation of specific binding to IL1RAP.

  • Assessment of blocking capacity for IL-1, IL-33, and IL-36 signaling.

  • Evaluation of downstream effects on cellular phenotypes.

• Production and Quality Control:

  • Expression in mammalian systems (e.g., ExpiCHO cells) to ensure proper folding and glycosylation.

  • Purification to >90% purity and confirmation by SEC and SDS-PAGE .

Successful antibodies have demonstrated efficacy through multiple mechanisms, including signaling blockade and immune-mediated elimination of target cells .

What are emerging areas of IL1RAP research beyond its known functions?

Several cutting-edge research directions are expanding our understanding of IL1RAP:

• Novel Signaling Partnerships:

  • Beyond IL-1 family receptors, IL1RAP interacts with receptor tyrosine kinases (FLT3, c-KIT) and potentially other signaling molecules .

  • Investigation of these non-canonical interactions may reveal new therapeutic opportunities.

• Tissue-Specific Functions:

  • Neuronal IL1RAP (IL-1RAPb isoform) interacts trans-synaptically with PTP sigma and can induce excitatory pre- and post-synaptic development .

  • The role of IL1RAP in tissue-specific inflammatory responses is an emerging area of investigation.

• Biomarker Development:

  • The QSOX1/IL1RAP ratio as a biomarker for NAFLD severity represents a new direction in using IL1RAP in diagnostic applications .

  • Investigation of IL1RAP as part of multi-parameter biomarker panels for disease detection and monitoring.

• Combination Therapies:

  • Exploration of synergistic effects between IL1RAP targeting and other therapeutic modalities (e.g., tyrosine kinase inhibitors, immune checkpoint inhibitors).

  • Development of dual-targeting approaches to address multiple pathways simultaneously.

• Structural Biology:

  • Detailed structural characterization of IL1RAP interactions with various binding partners to facilitate structure-based drug design.

  • Investigation of conformational changes induced by ligand binding and receptor complex formation.

These emerging areas represent promising directions for researchers seeking to expand the frontiers of IL1RAP biology and therapeutic applications.

How can multi-omics approaches enhance understanding of IL1RAP biology?

Integration of multiple omics approaches provides comprehensive insights into IL1RAP biology:

• Transcriptomics-Proteomics Integration:

  • Correlation of IL1RAP transcript and protein levels to identify post-transcriptional regulation mechanisms.

  • Analysis of gene expression patterns associated with high versus low IL1RAP expression .

• Spatial Transcriptomics:

  • Assessment of IL1RAP expression and associated gene expression changes in tissue context.

  • Spatial transcriptomic analysis revealed reduced canonical IL-1 signaling and chemokine expression in cardiac immune foci in IL1RAP-blocked mice .

• Phosphoproteomics:

  • Characterization of phosphorylation cascades downstream of IL1RAP activation to map signaling networks.

  • Identification of novel phosphorylation targets affected by IL1RAP modulation.

• Single-Cell Analysis:

  • Single-cell RNA sequencing of human tissues (e.g., atherosclerotic plaques) reveals expression patterns of IL1RAP and related cytokines/receptors at cellular resolution .

  • Identification of cell populations differentially expressing IL1RAP and responsive to IL1RAP-mediated signaling.

• Systems Biology Approaches:

  • Network analysis to identify hub genes and pathways connected to IL1RAP function.

  • Mathematical modeling of IL1RAP-mediated signaling dynamics across different cellular contexts.