IL13RA2 Human, Sf9

What is IL13RA2 and what characterizes its molecular structure?

IL13RA2 is a member of the type I cytokine receptor family, Type 5 subfamily. The human recombinant version produced in Sf9 insect cells is a single, glycosylated polypeptide chain containing 559 amino acids with a molecular mass of 64.3kDa, though it appears as 70-100kDa on SDS-PAGE due to glycosylation patterns . The protein contains residues 27-343 of the native sequence, often with additional tags (such as hIgG-His) at the C-terminus to facilitate purification.

IL13RA2 is also known by several synonyms, including CD213A2, CT19, IL-13R, IL13BP, and Interleukin-13-binding protein . Unlike many receptors, IL13RA2 lacks a functional cytoplasmic signaling domain, which significantly impacts its biological role.

How does IL13RA2 functionally differ from IL13RA1?

The critical functional distinction between these receptors lies in their signaling capabilities:

This difference is particularly important when developing targeted therapies. When generating IL13RA2-specific antibodies or nanobodies, researchers typically use IL13RA1 for deselection to ensure specificity, as demonstrated in CAR-T cell development approaches . The distinct functional profiles make IL13RA2 an attractive therapeutic target in certain cancers without disrupting normal IL-13 signaling through IL13RA1.

What is the significance of IL13RA2's role in disease pathology?

IL13RA2's role in disease appears to be context-dependent and sometimes contradictory across different cancer types. In hepatocellular carcinoma (HCC), TCGA database analysis revealed that IL13RA2 expression is higher in normal hepatic tissue compared to tumor tissue . Low expression of IL13RA2 in HCC correlates with poor patient survival, suggesting a potential tumor-suppressive role in this context .

Mechanistically, silencing of IL13RA2 in HCC promotes:

• Increased cell migration ability

• Enhanced cell proliferation (in HCCLM3 cells)

• Reduced apoptosis rates

• Partial epithelial-mesenchymal transition via increased ERK phosphorylation

Conversely, in glioblastoma and brainstem gliomas, IL13RA2 is often overexpressed and associated with more aggressive disease, making it an attractive therapeutic target in these contexts . This contextual variation highlights the importance of tissue-specific analysis when considering IL13RA2 as a therapeutic target.

What techniques provide the most comprehensive assessment of IL13RA2 expression?

Multiple complementary techniques should be employed for accurate IL13RA2 expression analysis:

Researchers should note that discrepancies between mRNA and protein levels are common with IL13RA2, as seen in MHCC97 cell lines where IL13RA2 was significantly overexpressed at the mRNA level but showed variable protein expression . This highlights the importance of using multiple detection methods.

How should researchers optimize recombinant IL13RA2 production in Sf9 cells?

Sf9 insect cells offer several advantages for IL13RA2 production, but require careful optimization:

Purification Strategy:

• Express with appropriate tags (e.g., hIgG-His tag) to facilitate purification

• Employ proprietary chromatographic techniques for isolation

• Aim for purity greater than 85.0% as determined by SDS-PAGE

Stability Considerations:

• Formulate in phosphate-buffered saline (pH 7.4) with 10% glycerol

• For long-term storage, add carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles

Quality Control Metrics:

• Verify molecular weight (approximately 64.3kDa core protein, appearing as 70-100kDa on SDS-PAGE due to glycosylation)

• Confirm glycosylation status, which is critical for proper folding and function

• Validate functional activity through binding assays with IL-13

These optimization strategies ensure production of high-quality IL13RA2 protein suitable for downstream applications, including antibody development, functional studies, and therapeutic targeting.

How does IL13RA2 silencing affect cellular signaling pathways?

IL13RA2 silencing induces significant changes in cellular signaling networks and behavior:

Primary Signaling Alterations:

• Increased extracellular signal-regulated kinase (ERK) phosphorylation

• Promotion of partial epithelial-mesenchymal transition (EMT)

Consequent Cellular Behavior Changes:

• Enhanced cell proliferation (observed in HCCLM3 but not MHCC97H, indicating cell-type dependence)

• Reduced apoptosis rates across multiple cell lines

• Increased migration ability (demonstrated in wound-healing and Transwell assays)

Interestingly, these effects appear independent of IL-13 addition, suggesting IL13RA2 may have intrinsic functions beyond its role as an IL-13 decoy receptor. The cell line-specific effects on proliferation suggest that IL13RA2's function may depend on the presence of other membrane proteins. In glioblastoma, for example, IL13RA2 interacts with mutant EGFR (EGFRvIII) to influence cell proliferation .

How can researchers develop highly specific IL13RA2-targeting molecules?

Developing highly specific IL13RA2-targeting molecules requires a strategic approach:

Development Pipeline:

• Immunization: Immunize alpacas with purified IL13RA2 recombinant protein

• Library Generation: Create phage display library enriched against IL13RA2

• Negative Selection: Use IL13RA1 as a deselection target to remove cross-reactive binders

• Deep Analysis: Perform next-generation sequencing (NGS) on the enriched library

• Computational Screening: Extract complementarity-determining regions (CDRs) and perform clustering analysis

• Biophysical Characterization: Determine EC₅₀ and thermal stability (Tₘ) of candidate binders

• Humanization: Modify selected binders to reduce immunogenicity while maintaining specificity

This approach has successfully yielded highly specific nanobodies, as demonstrated in the development of IL13RA2-targeting CAR-T cells where clone C1.1 emerged as a lead candidate following extensive screening and characterization .

What methodological approaches can resolve contradictory findings about IL13RA2 across cancer types?

Resolving the paradoxical roles of IL13RA2 in different cancers requires comprehensive methodological approaches:

Multi-level Expression Analysis:

• Compare mRNA expression with protein expression in the same samples

• Analyze expression in paired normal/tumor samples from the same patients

• Evaluate expression across large datasets while controlling for confounding variables

Context-specific Functional Studies:

• Examine IL13RA2 function in the presence/absence of key interaction partners (e.g., EGFRvIII)

• Assess function with/without exogenous IL-13 to distinguish ligand-dependent from intrinsic effects

• Compare effects in both traditional 2D culture and more physiologically relevant 3D models

Comprehensive Signaling Analysis:

• Map downstream signaling networks using phospho-proteomics

• Analyze effects on multiple pathways simultaneously (e.g., JAK/STAT, MAPK/ERK)

• Determine how IL13RA2 affects IL-13 signaling through IL13RA1/IL-4Rα complexes

This multi-faceted approach can help reconcile findings such as those in HCC, where IL13RA2 appears to have tumor-suppressive properties , versus gliomas, where it may promote malignancy .

What innovative strategies address challenges in IL13RA2-targeted cancer therapies?

Developing effective IL13RA2-targeted therapies faces several challenges, particularly for solid tumors. Recent innovations include:

Multi-functional "Armoured" CAR-T Cell Design:
Recent research has developed a novel IL13Rα2-targeted CAR-T construct incorporating multiple functional elements within a single retroviral vector:

• TGF-β receptor-blocking antibody to overcome immunosuppression

• Constitutively active GM-CSF intracellular receptor region for enhanced persistence

• Engineered single-chain IL-12 sequence for improved tumor cell killing

• Modified HER2 as a suicide switch for controlled cell elimination

Comparative Performance Metrics:
When compared to conventional CAR-T cells, these enhanced constructs demonstrated:

• Superior expansion and survival in nutrient-deprived environments

• Enhanced target cell elimination efficacy

• Maintained functionality in the presence of TGF-β

• Superior killing efficiency after prolonged exposure to tumor cells

Safety Mechanisms:
The incorporation of the HER2 module allows for regulation using FDA-approved drugs like Trastuzumab emtansine (T-DM1). In validation studies, cells exposed to T-DM1 lost their ability to kill target cells and secrete IFNγ, demonstrating effective regulation through this safety switch .

How should researchers evaluate IL13RA2-targeted therapies in experimental models?

Comprehensive evaluation of IL13RA2-targeted therapies requires multi-dimensional testing approaches:

In Vitro Testing Strategy:

• Standard Conditions: Basic cytotoxicity against IL13RA2-expressing target cells

• Stress Testing: Extended co-culture with target cells over multiple passages

• Microenvironment Simulation: Testing in the presence of:

  • TGF-β to model immunosuppression

  • Nutrient deprivation to mimic tumor core conditions

  • IL-13 to evaluate competitive binding effects

• Safety Evaluation: Confirm efficacy of suicide mechanisms (e.g., T-DM1 activation of HER2 module)

In Vivo Validation:

• Orthotopic Models: Use well-characterized tumor models with IL13RA2 expression (e.g., orthotopically injecting U87 cells modified to overexpress IL13RA2)

• Bioluminescence Monitoring: Group mice based on similar bioluminescence signals before treatment

• Multiple Endpoints: Assess tumor control, immune cell infiltration, and survival outcomes

This comprehensive testing strategy provides a more physiologically relevant assessment of therapeutic efficacy and helps bridge the gap between preclinical and clinical development.

What is the significance of IL13RA2 expression heterogeneity in developing personalized therapies?

The heterogeneous expression of IL13RA2 across and within cancer types has important implications for personalized therapy:

Expression Pattern Variations:

• Inter-Cancer Heterogeneity: Expression levels and prognostic significance vary dramatically between cancer types (e.g., lower in HCC tumors vs. normal tissue , higher in gliomas )

• Intra-Cancer Heterogeneity: Expression levels vary among different cell lines of the same cancer type (e.g., differences among HCC cell lines like MHCC97H, HCCLM3, and others )

• Functional Heterogeneity: The effect of IL13RA2 on cellular behavior varies depending on the presence of other receptors and signaling molecules

Implications for Therapy Development:

• Patient Stratification: Expression profiling can identify patients most likely to benefit from IL13RA2-targeted therapies

• Combination Approaches: Different expression patterns may require specific combination strategies

• Dynamic Monitoring: Expression changes during treatment may necessitate adaptive therapeutic approaches

Understanding this heterogeneity is crucial for developing truly personalized approaches to IL13RA2-targeted therapy, potentially explaining variable responses in clinical settings and guiding the design of more effective therapeutic strategies.

How can researchers leverage IL13RA2's unique structure for novel diagnostic applications?

The distinctive properties of IL13RA2 can be exploited for innovative diagnostic approaches:

Potential Diagnostic Applications:

• Liquid Biopsy Development: Detection of circulating IL13RA2 or IL13RA2-positive extracellular vesicles

• Molecular Imaging: Development of IL13RA2-targeted imaging agents using the highly specific nanobodies described in the literature

• Companion Diagnostics: Expression analysis to predict response to IL13RA2-targeted therapies

• Multi-marker Profiles: Combining IL13RA2 with other markers (like H3.3K27M, CD133, Ki67 in brainstem gliomas ) for improved diagnostic accuracy

Methodological Considerations:

• Prioritize specificity by using antibodies or nanobodies that don't cross-react with IL13RA1

• Validate across multiple sample types (tissue, serum, cerebrospinal fluid)

• Correlate expression with clinical outcomes to establish diagnostic utility

These approaches could transform IL13RA2 from a therapeutic target into a valuable diagnostic tool across multiple cancer contexts.

What are the most promising approaches for studying IL13RA2 protein-protein interactions?

Understanding IL13RA2's interactions with other proteins is crucial for elucidating its complex functions:

Advanced Methodological Approaches:

• Proximity Labeling Techniques: BioID or APEX2 fusion proteins to identify proteins in close proximity to IL13RA2 in living cells

• Cross-linking Mass Spectrometry: To capture transient or weak interactions

• Co-immunoprecipitation with Quantitative Proteomics: For unbiased identification of interaction partners

• Structural Biology Approaches: Cryo-EM or X-ray crystallography of IL13RA2 complexes with potential binding partners

• Live-cell Imaging: FRET or BiFC to visualize interactions in real-time

Priority Research Questions:

• How does IL13RA2 interact with EGFR variants in different cellular contexts?

• What membrane or intracellular proteins mediate IL13RA2's effects on ERK phosphorylation?

• Do post-translational modifications of IL13RA2 alter its interaction profile?

These approaches would help resolve the apparent contradictions in IL13RA2 function across different cancer types and potentially identify new therapeutic targets or combination strategies.