EPHB4 Antibody

What is EPHB4 and why is it significant in cancer research?

EPHB4 is a receptor tyrosine kinase belonging to the largest known family of receptor protein tyrosine kinases, the EPH receptors. This transmembrane protein (987 amino acids, 108.3 kDa) is highly expressed in placenta but also detected in kidney, liver, lung, pancreas, skeletal muscle, and heart . EPHB4 interacts primarily with its ligand ephrin-B2, mediating bidirectional signaling pathways that regulate cellular processes including adhesion, migration, and differentiation .

The significance of EPHB4 in cancer research stems from its complex, context-dependent roles. It's frequently overexpressed in multiple cancer types including breast, colon, bladder, endometrium, head and neck, prostate, and ovary . In acute myeloid leukemia (AML), high expression occurs in approximately 30% of cases . Interestingly, EPHB4 can function as both an oncogene and a tumor suppressor depending on the cancer type and stage. In some contexts, EPHB4 directly supports tumor cell survival by inhibiting apoptosis, while in others, particularly colorectal cancer, its loss correlates with worse patient outcomes .

What are the major types of EPHB4 antibodies available for research?

Research-grade EPHB4 antibodies fall into several categories:

Polyclonal antibodies:

• Examples include the H200 polyclonal antibody, raised against a 200 amino acid sequence spanning the cysteine-rich region and first fibronectin type III repeat of human EPHB4

• Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (AF3038), developed against the Leu16-Ala539 region

Monoclonal antibodies:

• MAb47 and MAb131 - novel monoclonal antibodies with different specificities. MAb131 targets fibronectin-like domain 1 of human EphB4, while MAb47 targets fibronectin-like domain 2 of both human and murine EphB4

• Mouse monoclonal IgG1 antibodies like the H-10 (sc-365510)

Domain-specific antibodies:

• Antibodies targeting specific domains such as the N-terminal region (N1N2)

• Antibodies targeting the cysteine-rich domain, which appears to be functionally important for ligand interaction

For reproducible research, investigators should select antibodies based on validated applications and appropriate species reactivity. The majority of commercially available antibodies recognize human EPHB4, though some cross-react with mouse, rat, or other species .

How can I validate the specificity of an EPHB4 antibody?

Validating antibody specificity is crucial for reliable research outcomes. Based on published methodologies, a comprehensive validation approach should include:

Knockout/knockdown controls:

• Use EPHB4 knockout cell lines as negative controls. Western blot analysis of parental and EPHB4 knockout HEK293T cells provides strong evidence of specificity, with bands at approximately 140 kDa in parental cells and absence in knockout cells

• RNA interference-mediated knockdown of EPHB4 can also serve as a control

Cross-reactivity testing:

• Test against related EPH family members using recombinant proteins. MAb47 and MAb131 were validated by testing binding to extracellular domains of different EphB receptors fused to alkaline phosphatase, confirming specificity only for EphB4

• Evaluate cross-reactivity with EphA family receptors using EphA-Fc proteins

Multiple detection methods:

• Compare results across different techniques (e.g., Western blot, immunoprecipitation, flow cytometry) to confirm consistent detection

• For example, the H200 antibody specificity was validated using Western blot, flow cytometry, and immunofluorescence in MCF10A cells with low endogenous EPHB4 versus cells engineered to overexpress EPHB4

Peptide competition assays:

• Pre-incubate antibody with specific peptides corresponding to the target epitope, which should abolish signal if the antibody is specific

• Research demonstrates that peptides from the cysteine-rich region successfully blocked H200 antibody function, confirming its epitope specificity

What are the optimal conditions for using EPHB4 antibodies in Western blotting?

Western blotting is one of the most common applications for EPHB4 antibodies. Based on published protocols, the following parameters yield optimal results:

Sample preparation:

• Extract total protein from cells or tissues using standard lysis buffers containing protease inhibitors

• For membrane protein enrichment, consider using membrane fraction isolation protocols

• Use reducing conditions for most applications (the native EphB4 protein appears at approximately 120-140 kDa)

Gel separation and transfer:

• 6% SDS-PAGE gels are recommended for optimal separation of the high molecular weight EphB4 protein

• Transfer to PVDF membranes (e.g., Immobilon-P) yields better results than nitrocellulose for this large protein

Antibody concentration and incubation:

• Primary antibody dilutions:

  • Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (AF3038): 2 μg/mL

  • H200 polyclonal antibody: 1:1000 dilution

• Incubation: Overnight at 4°C for optimal signal-to-noise ratio

Detection system:

• HRP-conjugated secondary antibodies with enhanced chemiluminescence detection systems work well

• For the H200 antibody, use of the Lumi-Light PLUS Western Blotting kit with 10-30 seconds exposure to ECL film has been successful

Positive controls:

• Cell lines known to express EphB4: K562 (human chronic myelogenous leukemia), COLO 205 (colorectal adenocarcinoma), ZR-75 (breast cancer), HUVEC (human umbilical vein endothelial cells), and MCF-7 (breast cancer)

Internal loading controls:

• GAPDH is commonly used as a loading control when examining EphB4 expression

What protocols are recommended for immunohistochemical detection of EPHB4?

Immunohistochemistry (IHC) is valuable for visualizing EPHB4 distribution in tissues. Based on published methods, the following protocol has been validated:

Tissue preparation:

• Formalin-fixed, paraffin-embedded (FFPE) sections are suitable

• Optimal section thickness: 5-6 μm

Antigen retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Alternative: immersion fixed paraffin-embedded sections

Antibody concentration and incubation:

• Primary antibody:

  • Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody: 15 μg/mL

  • Incubation: Overnight at 4°C for optimal staining

Detection system:

• Anti-Goat HRP-DAB Cell & Tissue Staining Kit has been successfully used for visualization (brown staining)

• Counterstain with hematoxylin (blue) to identify nuclei

Controls and interpretation:

• Positive tissue controls: Human kidney expresses EPHB4 and serves as a good positive control

• Normal adjacent tissue as internal negative/low expression control

• EPHB4 localizes primarily to cell membranes of epithelial cells, with both apical and basal surfaces showing comparable intensity

Pattern interpretation:

• In colorectal tissues, EPHB4 shows strong staining in tumor epithelial cells (both absorptive surface epithelial cells and crypt mucus-secreting cells) with weak, diffuse staining in normal mucosa

• Look for membrane-localized staining pattern consistent with receptor tyrosine kinase localization

How should EPHB4 antibodies be optimized for flow cytometry applications?

Flow cytometry enables quantitative analysis of EPHB4 expression at the single-cell level. Based on published protocols, consider the following optimization steps:

Cell preparation:

• Single-cell suspensions are critical; avoid cell clumping

• For adherent cells, use enzymatic dissociation methods that preserve surface epitopes

• Fix cells in 2-4% paraformaldehyde if not analyzing immediately

Antibody selection and titration:

• Choose antibodies specifically validated for flow cytometry, such as the Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (AF3038)

• Perform antibody titration experiments to determine optimal concentration

• Starting dilution: Use manufacturer's recommendation, then optimize

Staining protocol:

• Standard protocol for MCF-7 breast cancer cells:

  • Incubate with primary antibody (e.g., AF3038)

  • Follow with fluorochrome-conjugated secondary antibody (e.g., Phycoerythrin-conjugated Anti-Goat IgG)

• Include appropriate isotype control (e.g., AB-108-C)

Controls:

• Positive cell line controls: MCF-7 human breast cancer cells express EPHB4 and serve as a good positive control

• Negative controls:

  • Isotype controls to assess non-specific binding

  • EPHB4 knockout cells when available

  • Secondary antibody only controls

Data analysis:

• Analyze shift in fluorescence compared to isotype control

• Report median fluorescence intensity (MFI) rather than percent positive when examining quantitative differences

• Consider using histogram overlays to visualize expression differences between samples

How does EPHB4 expression differ across cancer types, and what methods best detect these differences?

EPHB4 expression varies significantly across cancer types, with both overexpression and loss of expression observed depending on the context:

Cancer types with EPHB4 overexpression:

• Breast cancer: Frequently overexpressed, detected in cell lines like MCF-7, ZR-75

• Colon cancer: Increased expression in 82% of tumor samples compared to matched normal tissue

• Acute myeloid leukemia (AML): Highly expressed in approximately 30% of cases

• Other cancers with documented overexpression: bladder, endometrium, head and neck, prostate, and ovary

Cancer types with reduced EPHB4 expression:

• Subset of colorectal cancers: Loss of EPHB4 correlates with poorer prognosis in some studies

• Mechanism of loss: EPHB4 promoter hypermethylation in some colorectal tumors

Detection methodologies comparison:

For comprehensive analysis of EPHB4 in cancer research, a multi-method approach is recommended. Quantitative RT-PCR analysis has successfully demonstrated increased expression in 82% of colorectal tumor samples compared to matched normal tissue . This can be validated at the protein level using Western blot, with subsequent immunohistochemistry to determine cellular localization and heterogeneity within tumor samples.

What is the prognostic significance of EPHB4 expression in different cancers?

The prognostic significance of EPHB4 varies by cancer type, highlighting its context-dependent roles:

Colorectal cancer:

• Patients with low EPHB4 tumor levels had significantly shorter survival than patients with high EPHB4 expression (median survival 1.8 years versus >9 years)

• This finding was validated in an independent set of 125 tumor samples

• EPHB4 promoter hypermethylation correlates with reduced expression and worse outcomes

Acute myeloid leukemia (AML):

• EPHB4 drives survival in a subset of AML cases

• High expression correlates with poor outcomes in some studies

• EPHB4 promotes leukemia survival via AKT activation

Prostate cancer:

• EPHB4 expression is induced in PTEN-null prostate cancer

• Contributes significantly to tumor initiation

• Continues to promote tumor progression in castration-resistant prostate cancer

Methodological considerations for prognostic studies:

• Use of tissue microarrays allows high-throughput analysis of EPHB4 expression across large cohorts

• Kaplan-Meier survival analysis with log-rank test is the standard statistical approach for correlating EPHB4 expression with patient outcomes

• Multivariable analysis should be performed to determine if EPHB4 is an independent prognostic factor

• Cutoff determination for "high" versus "low" expression should be carefully justified

Researchers should recognize that depending on cancer type and context, EPHB4 may function as either a tumor suppressor or oncogene, which explains the seemingly contradictory prognostic associations across different malignancies.

How do EPHB4 antibodies contribute to understanding cancer cell signaling mechanisms?

EPHB4 antibodies have been instrumental in elucidating signaling pathways in cancer, revealing complex interactions:

PI3K/AKT signaling:

• Knockdown of EPHB4 inhibits PI3K/AKT signaling in AML cells

• This is accompanied by a reduction in cell viability, which can be rescued by constitutively active AKT

• EPHB4 antibodies can be used to monitor AKT phosphorylation status following EPHB4 modulation

Ligand-dependent versus ligand-independent signaling:

• Antibodies targeting specific epitopes have revealed that:

  • Ligand-independent signaling promotes tumor growth and survival

  • Ligand-dependent signaling (EphB4-ephrinB2) can be tumor suppressive

• H200 antibody (targeting cysteine-rich region) causes phosphorylation followed by degradation of EPHB4 protein, suggesting a ligand-mimetic mechanism

Receptor internalization and degradation mechanisms:

• Treatment with certain antibodies (e.g., H200) leads to downregulation of both EPHB4 gene expression and protein levels in cancer cells

• Western blot analysis following antibody treatment shows time-dependent reduction in EPHB4 protein levels (24h, 48h, 72h)

Methodological approaches to study signaling:

• Phosphorylation status assessment:

  • Western blot with phospho-specific antibodies following EPHB4 antibody treatment

  • Immunoprecipitation of EPHB4 followed by phosphotyrosine detection

• Pathway analysis:

  • Combined use of EPHB4 antibodies with inhibitors of downstream pathways

  • Phospho-protein arrays to identify activated pathways

• Temporal dynamics:

  • Time-course experiments to distinguish immediate versus delayed effects

  • Pulse-chase experiments to follow receptor trafficking

Researchers can leverage these approaches to determine whether an EPHB4 antibody activates or inhibits signaling, and which downstream pathways are affected in specific cancer contexts.

What mechanisms underlie the anti-tumor effects of therapeutic EPHB4 antibodies?

EPHB4 antibodies exhibit anti-tumor activity through multiple mechanisms:

Receptor degradation:

• H200 polyclonal antibody treatment causes EPHB4 protein degradation in cancer cells

• After 72 hours of treatment, Western blot analysis shows significant reduction in EPHB4 protein levels compared to both untreated cells and IgG control-treated cells

• This correlates with reduced expression of the EPHB4 gene, suggesting feedback regulation

Ligand-mimetic activity:

• Some antibodies, like H200, appear to mimic the effect of ephrin-B2 ligand binding

• This induces phosphorylation of EPHB4, followed by receptor internalization and degradation

• This mechanism potentially converts tumor-promoting ligand-independent signaling to tumor-suppressive ligand-dependent signaling

Direct induction of cell death:

• Treatment with H200 antibody causes detachment of confluent cancer cell monolayers after 24 hours

• By 72 hours, >80% of cells stain with trypan blue, indicating cell death

• These effects are antibody-specific, as not all anti-EPHB4 antibodies induce this response

Inhibition of specific domains:

• MAb131 targets fibronectin-like domain 1 of human EPHB4 and inhibits tumor cells expressing EPHB4 in vitro

• MAb47 targets fibronectin-like domain 2 of both human and murine EPHB4 and inhibits angiogenesis

Combination therapy enhancement:

• Combination of MAb47 and bevacizumab enhances antitumor activity and induces tumor regression

• This suggests potential synergy with anti-angiogenic therapies

The diversity of mechanisms suggests that different epitope-targeting antibodies may have distinct therapeutic applications depending on cancer type and molecular context.

How can researchers evaluate the efficacy of EPHB4 antibodies in preclinical cancer models?

Evaluating EPHB4 antibody efficacy requires robust preclinical models and appropriate endpoints:

In vitro models and assays:

In vivo models:

• Subcutaneous xenograft models: Used to demonstrate that MAb47 inhibits growth of both EPHB4-positive and EPHB4-negative tumors

• Orthotopic models: More relevant microenvironment

• Patient-derived xenografts: Better recapitulate tumor heterogeneity

• Metastasis models: Evaluate effects on tumor spread

Dosing considerations:

• H200 antibody dilutions ranging from 1/100 to 1/10,000 have been tested in vitro, with concentration-dependent effects

• For the humanized antibody hAb47, careful dose-response studies should be conducted to determine optimal dosing in vivo

Molecular and cellular assessments:

• Receptor downregulation: Measure EPHB4 protein levels by Western blot at multiple timepoints

• Signaling inhibition: Assess phosphorylation of AKT and other downstream targets

• Angiogenesis inhibition: CD31 staining of tumor vessels, microvessel density quantification

Combination approaches:

• Evaluate synergy with standard chemotherapeutics

• Test combinations with targeted therapies (e.g., bevacizumab enhanced MAb47 efficacy)

• Explore potential with immune checkpoint inhibitors

Researchers should select models based on the cancer type and expected mechanism of action. For example, MAb131 showed efficacy against AML in vitro and in vivo, making it a promising candidate for hematologic malignancies .

What are the key considerations when developing EPHB4 antibodies for clinical applications?

Development of EPHB4 antibodies for clinical use requires careful consideration of several factors:

Epitope selection:

• Target domains with functional significance:

  • Cysteine-rich region appears to be a potential ligand interacting interface

  • Fibronectin-like domains have distinct functional roles

• Consider cross-reactivity with other EPH family members (high sequence homology)

• Select epitopes conserved between human and mouse for better translational studies

Antibody format optimization:

• Humanization: MAb47 and MAb131 have been humanized (hAb47 and hAb131) while maintaining similar affinity for EPHB4 and efficacy

• IgG subclass selection affects effector functions and half-life

• Consider alternative formats (F(ab')2, bispecific antibodies) based on mechanism

Pharmacological considerations:

• Binding affinity measurements using surface plasmon resonance or bio-layer interferometry

• Pharmacokinetic studies to determine half-life and tissue distribution

• Potential immunogenicity assessment

Context-dependent effects:

• Biomarker development to identify responsive patient populations

• EPHB4 expression levels may predict response

• Consider potential differential effects in different cancer types (tumor suppressor vs. oncogene)

Antibody quality and manufacturing:

• Develop robust potency assays based on mechanism of action

• Ensure consistent glycosylation pattern if effector function is important

• Stability studies under various storage conditions

Combination strategies:

• Identify synergistic combinations (e.g., MAb47 with bevacizumab)

• Understand potential interactions with standard-of-care therapies

• Develop rationale for combination approaches based on pathway analysis

The dual nature of EPHB4 as both potential tumor promoter and suppressor highlights the importance of careful antibody development with thorough understanding of the target biology in specific cancer contexts.

How do post-translational modifications affect EPHB4 detection and function?

Post-translational modifications (PTMs) significantly impact EPHB4 biology and detection methods:

Key EPHB4 post-translational modifications:

• Phosphorylation: Critical for receptor activation and signaling

• Glycosylation: EPHB4 is known to undergo N-linked glycosylation

• Ubiquitination: Involved in receptor degradation pathways

• Proteolytic processing: May generate truncated forms

Impact on antibody detection:

• Western blot analysis often reveals multiple bands:

  • The predicted full-length EphB4 protein appears at approximately 120-140 kDa

  • Lower molecular weight bands may represent proteolytic fragments or alternative isoforms

• Deglycosylation treatments prior to Western blot can help distinguish glycosylation-dependent size variations

• Phosphorylation-specific antibodies can detect activated EPHB4

Methodological considerations:

• Sample preparation should preserve PTMs of interest (phosphatase inhibitors for phosphorylation studies)

• For glycosylation studies, compare results with and without PNGase F treatment

• When studying receptor degradation, include proteasome inhibitors (MG132) or lysosomal inhibitors (chloroquine) to determine degradation pathway

Functional consequences:

• Antibody binding to specific domains may affect particular PTMs

• H200 antibody treatment causes phosphorylation and subsequent degradation of EPHB4

• Understanding the relationship between antibody binding and PTM induction can inform therapeutic development

Researchers investigating EPHB4 PTMs should carefully select antibodies that recognize the protein regardless of modification status or choose modification-specific antibodies depending on the research question.

How do ligand-dependent and ligand-independent EPHB4 signaling differ in cancer contexts?

The dichotomy between ligand-dependent and ligand-independent EPHB4 signaling is crucial for understanding its complex roles in cancer:

Ligand-dependent signaling:

• Activated by interaction with ephrin-B2 on adjacent cells

• Generally tumor suppressive in many contexts

• Promotes cell adhesion and organized tissue architecture

• In colorectal cancer, may maintain the differentiated state of tumor cells

Ligand-independent signaling:

• Occurs when EPHB4 is overexpressed without corresponding ligand engagement

• Typically tumor promoting

• Supports cell survival, migration, and invasion

• Activates PI3K/AKT pathway in AML cells

Experimental approaches to distinguish signaling modes:

Antibody-based modulation:

• Some antibodies (e.g., H200) appear to convert ligand-independent to ligand-dependent-like signaling

• These antibodies bind the cysteine-rich region, a potential ligand interacting interface

• This conversion results in receptor phosphorylation, internalization, and degradation

Cancer context relevance:

• In AML, EPHB4 promotes cell survival via AKT activation in a likely ligand-independent manner

• In colorectal cancer, loss of EPHB4 expression correlates with worse prognosis, suggesting tumor-suppressive ligand-dependent signaling is dominant

• In prostate cancer, EphB4-ephrin-B2 interaction contributes to tumor initiation and progression to castration resistance

Understanding the switch between these signaling modes offers opportunities for therapeutic intervention, particularly with antibodies that can shift the balance toward tumor-suppressive signaling.

What emerging technologies are advancing EPHB4 antibody research?

Several cutting-edge technologies are transforming EPHB4 antibody research:

Single-cell analysis technologies:

• Single-cell RNA sequencing to identify heterogeneous EPHB4 expression within tumors

• Mass cytometry (CyTOF) with EPHB4 antibodies to correlate expression with multiple markers

• Imaging mass cytometry for spatial information while maintaining single-cell resolution

Advanced imaging techniques:

• Super-resolution microscopy to visualize EPHB4 clustering and co-localization

• Intravital microscopy using fluorescently labeled antibodies to track EPHB4 dynamics in vivo

• Förster resonance energy transfer (FRET) to detect EPHB4-ephrin-B2 interactions in real-time

Antibody engineering approaches:

• Nanobodies against EPHB4 for improved tissue penetration

• Bispecific antibodies targeting EPHB4 and immune effector cells

• Antibody-drug conjugates for targeted delivery of cytotoxic agents

High-throughput functional screening:

• CRISPR-Cas9 screens to identify synthetic lethal interactions with EPHB4

• Combinatorial antibody library screening against specific EPHB4 domains

• Automated high-content imaging to assess antibody effects on cellular phenotypes

Molecular dynamics simulations:

• In silico modeling of antibody-epitope interactions

• Structure-based design of antibodies with improved binding or functional properties

• Prediction of conformational changes induced by antibody binding

Clinical translation technologies:

• Liquid biopsy techniques to detect EPHB4 expression in circulating tumor cells

• Companion diagnostic development for patient stratification

• Radiolabeled antibodies for PET imaging (e.g., evaluation of EphB4 as target for image-guided surgery of breast cancer )

Researchers can leverage these technologies to gain deeper insights into EPHB4 biology and develop more effective therapeutic antibodies with improved target engagement and functional outcomes.