ERBB3 Antibody

What is the functional significance of ERBB3 in cancer progression?

ERBB3 (HER3) is a tyrosine-protein kinase receptor that plays an essential role as a cell surface receptor for neuregulins. Despite having a pseudokinase domain with limited catalytic activity, ERBB3 is a critical signaling node in cancer progression through several mechanisms:

• ERBB3 contains six binding sites for the regulatory subunit p85 of phosphoinositide-3-kinase, making it one of the most potent known activators of the PI3K/AKT signaling pathway

• Upon ligand binding (primarily neuregulin-1/NRG1), ERBB3 undergoes phosphorylation on tyrosine residues, promoting its association with the p85 subunit of PI3K

• ERBB3 mediates resistance to therapeutics targeting other ErbB family members, particularly EGFR and HER2-directed therapies

• High expression of ERBB3 correlates with poor prognosis in several cancer types, including lung adenocarcinoma, breast cancer, colorectal cancer, and ovarian cancer

For experimental assessment of ERBB3's role in cancer, researchers should consider both ligand-dependent activation (using NRG1) and ligand-independent mechanisms through heterodimer formation with other ErbB receptors, particularly in HER2-overexpressing models .

How do different types of ERBB3 antibodies compare in research applications?

Different ERBB3 antibodies vary in their binding domains, mechanisms of action, and research applications:

When selecting an antibody for research, consider whether you're studying:

• Total ERBB3 expression (use C-terminal antibodies)

• Receptor activation (use domain-specific antibodies or phospho-specific antibodies)

• ERBB3-mediated resistance mechanisms (use antibodies that block both ligand-dependent and independent activation)

What are the recommended protocols for detecting ERBB3 in different experimental systems?

Detection methods for ERBB3 vary by experimental approach:

Western Blotting:

• Use reducing conditions with appropriate buffer systems

• ERBB3 typically appears at approximately 185 kDa

• Recommended antibody dilutions: 1 μg/mL for monoclonal antibodies

• Positive controls: MDA-MB-453 and MCF-7 breast cancer cell lines

Immunoprecipitation:

• Several antibodies show good efficiency (ab34641, ab20162)

• LNCaP prostate cancer cells can serve as a suitable model

• Include both untreated and ligand-stimulated samples to assess activation status

Flow Cytometry:

• Multiple conjugated antibodies available (Alexa Fluor 647, PE, FITC)

• Careful titration required; check specific antibody recommendations

• Consider membrane permeabilization for total vs. surface ERBB3 assessment

Immunofluorescence:

• Antibodies such as sc-7390 have been validated for IF applications

• Include co-staining for dimerization partners (EGFR, HER2) for interaction studies

When troubleshooting detection issues, consider cell-specific expression levels, receptor internalization following ligand treatment, and the need for enrichment methods in low-expressing systems.

How can researchers effectively study ERBB3-mediated resistance to targeted therapies?

ERBB3 plays a crucial role in resistance to EGFR and HER2-directed therapies. To study this phenomenon:

Experimental Design Approaches:

• Develop resistant cell lines through long-term exposure to EGFR/HER2 inhibitors

• Compare ERBB3 phosphorylation status between parental and resistant lines

• Perform transient ERBB3 knockdown to assess reversibility of resistance

• Combine ERBB3-targeting antibodies with EGFR/HER2 inhibitors to evaluate synergy

Key Methodological Considerations:

• Include assessments of PI3K/AKT pathway activation, as this is a primary mechanism of ERBB3-mediated resistance

• Monitor recovery of ERBB3 phosphorylation over time after inhibitor treatment

• In pancreatic cancer models, ERBB3 knockdown has been shown to convert previously Erlotinib-sensitive cells to resistant ones

• For HER2-overexpressing models, assess both ligand-dependent and ligand-independent activation of ERBB3

The experiment by Liles et al. provides an important framework, demonstrating that "pancreatic cancer cell lines that are initially sensitive to Erlotinib become resistant upon transient knock down of ERBB3" , which challenges conventional models and warrants further investigation.

What methodologies are most effective for evaluating ERBB3 antibodies that lock the receptor in an inactive conformation?

Several therapeutic antibodies function by locking ERBB3 in its tethered, inactive conformation. To evaluate such antibodies:

Structural Analysis:

• X-ray crystallography of antibody Fab fragments bound to ERBB3 extracellular domain reveals binding epitopes and conformational effects

• The crystal structure of KTN3379 Fab bound to ERBB3 extracellular domain demonstrated binding at the domain 2-3 boundary, preventing conformational rearrangement

Functional Assays:

• NRG binding inhibition: Measure whether antibodies block fluorescently labeled NRG binding to cells expressing ERBB3

  • Example protocol: Treat ERBB3-expressing cells (e.g., Ba/F3) with 10 nM antibody for 1 hour at 4°C, followed by increasing concentrations of fluorescently labeled NRG for 2 hours at 4°C

• Conformational stability: Assess whether antibodies prevent conformational changes even in high ligand concentrations

• Dimerization assays: Evaluate antibody effects on ERBB3 heterodimerization with EGFR or HER2

Binding Kinetics Assessment:

• Surface Plasmon Resonance (SPR) to measure binding kinetics of antibodies to the full extracellular domain versus individual domains

• KTN3379 shows picomolar affinity with extremely slow off-rates, a desirable property for therapeutic antibodies

• Compare binding to tethered versus extended conformations of ERBB3

What are the experimental methods to distinguish between ligand-dependent and ligand-independent ERBB3 activation?

Distinguishing between these activation mechanisms is crucial for comprehensive ERBB3 research:

For Ligand-Dependent Activation:

• Use neuregulin-1 (NRG1) at 20 ng/ml as a standard stimulation condition

• Pre-incubation with NRG1 for 48 hours can up-regulate ERBB3 expression and activity in experimental systems

• Monitor phosphorylation of specific tyrosine residues in ERBB3 that promote association with PI3K

• Use domain 1-targeting antibodies that specifically block ligand binding (e.g., Lumretuzumab)

For Ligand-Independent Activation:

• Study ERBB3 in HER2-overexpressing models (without adding NRG1)

• The Ba/F3 cell model expressing ERBB2 V956R can be used to study ligand-independent transactivation of ERBB3

• Assess cell survival in IL-3-independent conditions without NRG1 addition

• Use antibodies that lock ERBB3 in inactive conformation (e.g., KTN3379, LJM716) which block both activation mechanisms

Comparative Analysis:

• Design experiments with four conditions: untreated, NRG1 only, HER2 overexpression only, and combined NRG1 with HER2 overexpression

• Compare phosphorylation patterns and downstream signaling outcomes

• Use selective inhibitors to dissect pathway dependencies

How can researchers design experiments to evaluate trans-activating mutations of ERBB3?

Recent studies have identified activating mutations in ERBB3 that can drive oncogenic signaling. To study these:

Functional Genetics Screening:

• The Ba/F3 cell system provides a valuable model for screening ERBB3 mutations

• Ba/F3 cells are dependent on IL-3 for survival, but this dependency can be overcome by expression of activating RTK mutations

• Design an experimental workflow:

  • Generate a mutation library in ERBB3 cDNA

  • Transduce into Ba/F3 cells expressing ERBB2 V956R

  • Select for cells surviving in the absence of both IL-3 and NRG-1

  • Extract genomic DNA from surviving populations to identify transforming mutations

Validation of Identified Mutations:

• Recreate individual mutations using site-directed mutagenesis

• Test their transforming ability in multiple cell models

• Evaluate sensitivity to different ERBB3 antibodies and ERBB family tyrosine kinase inhibitors

• Assess whether mutations affect antibody binding, particularly for antibodies targeting specific domains

Structural Analysis:

• Determine how mutations affect receptor conformation and dimerization

• Evaluate whether mutations preferentially promote interactions with specific dimerization partners

• Assess impact on binding of different classes of therapeutic antibodies

What are the best approaches for developing antibodies targeting the domain 2-3 hinge region of ErbB receptors?

The domain 2-3 hinge region represents a valuable therapeutic target across multiple ErbB family members:

Structural Considerations:

Engineering Approaches:

• Structure-guided modifications to existing antibodies:

  • Conservative modifications in the VH CDR2 may alleviate clashing with domain 3 loops

  • Interface-optimizing mutations can enhance binding affinity

  • These modifications could potentially convert ERBB3-specific antibodies to target EGFR or ERBB4

Validation Methods:

• Binding kinetics analysis using SPR to evaluate on/off rates

• Cell-based assays to confirm inhibition of receptor activation

• Comparative analysis across multiple ErbB family members to assess selectivity

• Assessment of ability to block both ligand-dependent and ligand-independent activation

This approach offers a potential advantage over direct competition with ligand binding, as targeting an allosteric site essential for receptor activation can provide improved binding kinetics and versatile mechanisms of action .