EPHA10 Antibody, Biotin conjugated

What is EPHA10 and why is it significant in cancer research?

EPHA10 (Ephrin receptor A10) is a receptor tyrosine kinase that shows minimal expression in normal tissues (primarily restricted to male testis) but is highly expressed in several malignancies. Its significance stems from its correlation with tumor progression and poor prognosis in several cancer types, particularly triple-negative breast cancer (TNBC). The limited expression in normal tissues coupled with high expression in tumors makes it an attractive therapeutic target with potentially minimal adverse effects . Research has shown that EPHA10 expression is significantly correlated with lymph node metastasis and higher tumor stage in human breast cancer specimens, suggesting its role as a potential driver of malignant phenotypes .

In which cancer types has EPHA10 overexpression been documented?

EPHA10 has been found to be highly expressed in tumor regions of breast cancer (particularly TNBC), lung cancer, and ovarian cancer. The expression is not limited to the cancer cells themselves but extends to cells in the tumor microenvironment, specifically immunosuppressive myeloid cells such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) . These cell populations are known to suppress antitumor immunity, suggesting that EPHA10 may play a role in immune evasion mechanisms employed by tumors .

How does EPHA10 relate to the broader Ephrin receptor family?

EPHA10 shares a similar architecture with other EphA family members (EphA1-EphA8) but has distinct functions and expression patterns. Unlike other EphA receptors that are broadly expressed in various tissues, EPHA10 exhibits a highly restricted expression pattern, making it particularly valuable as a cancer-specific target . Studies using ELISA-based methods have confirmed that anti-EPHA10 antibodies can bind specifically to EPHA10 without cross-reactivity to other EphA family members, highlighting the structural uniqueness of EPHA10 despite architectural similarities .

What are the most reliable techniques for detecting EPHA10 expression in tissue samples?

Several complementary techniques have proven effective for detecting EPHA10 expression:

• Immunohistochemistry (IHC): Used for assessing EPHA10 expression in human cancer specimens, allowing correlation with clinicopathological features like lymph node metastasis and tumor stage .

• Multiplex immunofluorescence: This technique has been used to visualize co-localization of EPHA10 with specific cell populations in the tumor microenvironment. For example, studies have shown EPHA10 co-localization with TAMs (F4/80+ CD163+) and MDSCs (CD11b+ Gr-1+) but not with tumor-infiltrating T cells (CD3+) .

• Flow cytometry: This method evaluates cell surface expression of EPHA10 on both cancer cells and immune cell populations, allowing quantitative assessment of expression levels .

• ELISA: Enables quantitative detection of EPHA10 in human serum, plasma, cell culture supernatant, and other biological samples .

For optimal results, researchers should consider using multiple detection methods to confirm expression patterns.

How can EPHA10 antibody specificity be validated experimentally?

Validating EPHA10 antibody specificity involves several critical steps:

• Binding specificity assessment: Using plate-based ELISA to evaluate binding affinity to EPHA10 versus other EphA family members (EphA1-EphA8) .

• Cell surface recognition validation: Flow cytometry comparing cells expressing EPHA10 (e.g., BT-549 expressing human EPHA10) versus matched control cells (mock transfected) .

• Immunofluorescence microscopy: Confirming the ability to recognize intact EPHA10 structure on the cell surface .

• Cross-species reactivity testing: If applicable, verifying recognition of both human and mouse EPHA10, which share 91% amino acid identity .

• Kinetic binding analysis: Using techniques like octet analysis to determine binding stability, association, and dissociation rates at serial antigen concentrations .

These validation steps ensure that experimental results genuinely reflect EPHA10-specific biology rather than cross-reactivity with related proteins.

What approaches have been successful in generating specific anti-EPHA10 monoclonal antibodies?

The generation of highly specific anti-EPHA10 monoclonal antibodies has been achieved through a strategic approach:

• Initial immunization with purified human EPHA10 extracellular domain (ECD)-Fc fusion protein to provoke an immune response .

• Booster immunization with whole cells expressing human EPHA10, preserving the native conformation and modifications of the antigen during antibody selection .

• Hybridoma technology to fuse responding B cells with immortalized myeloma cells, producing hybridoma clones that continuously secrete anti-EPHA10 antibodies .

• Multi-stage screening process:

  • First screening: Selection for recognition of EPHA10 ECD-Fc but not Fc alone

  • Subsequent screenings: Biological activity assays including flow cytometry, immunofluorescence, ELISA, and in vivo antibody tracking

This comprehensive approach has yielded antibodies with high specificity and favorable pharmacokinetic properties.

What parameters should be evaluated when characterizing a new anti-EPHA10 antibody?

When characterizing a new anti-EPHA10 antibody, researchers should evaluate:

• Specificity: Binding to EPHA10 without cross-reactivity to other EphA family members (EphA1-EphA8) .

• Binding affinity: Quantitative assessment of antibody-antigen interaction strength and kinetics .

• Recognition of native conformation: Ability to bind cell surface-expressed EPHA10 in its natural conformation .

• Pharmacokinetics and biodistribution: Half-life in circulation and specific accumulation in EPHA10-expressing tissues versus other organs .

• Functional effects: Impact on tumor growth, immune cell activation, or other relevant biological endpoints .

• Cross-species reactivity: Recognition of EPHA10 from different species, especially if planning preclinical studies in animal models .

Comprehensive characterization across these parameters enables informed selection of the most promising antibody candidates for specific research applications.

How does biotin conjugation affect the functionality of EPHA10 antibodies?

Biotin conjugation provides several advantages for EPHA10 antibody applications while potentially influencing functionality in specific ways:

• Detection sensitivity: Biotin conjugation enables signal amplification through streptavidin-based detection systems, enhancing sensitivity for low-abundance EPHA10 detection in tissues or cells.

• Epitope accessibility: The biotin moiety size and conjugation chemistry may affect antibody binding to certain EPHA10 epitopes, particularly if conjugation occurs near the antigen-binding region.

• Pharmacokinetics: Biotin conjugation may alter the in vivo half-life and tissue distribution properties of EPHA10 antibodies, which should be experimentally verified if transitioning from unconjugated to conjugated versions.

• Versatility in detection methods: Biotin-conjugated antibodies can be detected using various streptavidin-coupled reporters (HRP, fluorophores, gold particles), allowing flexibility in experimental design.

Researchers should validate that biotin conjugation does not compromise the specificity or affinity of the EPHA10 antibody through appropriate control experiments comparing conjugated and unconjugated versions.

How can EPHA10 antibodies be utilized in cancer immunotherapy research?

EPHA10 antibodies have demonstrated significant potential in cancer immunotherapy research through several applications:

• Direct tumor targeting: Anti-EPHA10 monoclonal antibodies specifically accumulate in tumor regions expressing EPHA10 without significant accumulation in other organs, making them valuable for targeted therapy approaches .

• Enhanced antitumor immunity: Treatment with anti-EPHA10 antibodies (particularly clone #4) has been shown to:

  • Impair tumor growth in syngeneic TNBC mouse models

  • Improve therapeutic response rates

  • Enhance CD8+ T cell-mediated antitumor immunity

• Combination therapy studies: EPHA10 antibodies can be investigated in combination with other immunotherapeutic agents, particularly given the association between EPHA10 expression and PD-L1 expression and immunosuppression .

• CAR-T cell development: Anti-EPHA10 antibodies have been used to develop EPHA10-specific chimeric antigen receptor T cells that significantly inhibited TNBC cell viability in vitro and tumor growth in vivo .

These applications highlight the versatility of EPHA10 antibodies in advancing cancer immunotherapy research, particularly for TNBC, which has limited targeted therapy options.

What protocols are recommended for EPHA10 detection in clinical samples?

For reliable EPHA10 detection in clinical samples, the following protocols are recommended:

• ELISA-based detection in serum/plasma:

  • Validated ELISA kits are available for quantitative determination of EPHA10 in human serum, plasma, and other biological samples

  • For plasma samples, EDTA or citrate anticoagulants are recommended rather than heparin

  • Sample processing should occur promptly following collection to minimize protein degradation

• Immunohistochemistry for tissue sections:

  • Formalin-fixed paraffin-embedded (FFPE) sections are typically used

  • Antigen retrieval steps are critical for optimal EPHA10 detection

  • Counterstaining with markers for specific cell populations (e.g., CD3 for T cells, F4/80 and CD163 for TAMs, CD11b and Gr-1 for MDSCs) enables assessment of EPHA10 expression across different cell types

• Flow cytometry for circulating and tumor-infiltrating cells:

  • Fresh samples yield optimal results

  • Multi-parameter panels including EPHA10 and lineage markers provide the most informative data

  • Controls using cells known to express or lack EPHA10 should be included in each experiment

Whichever method is selected, appropriate controls and standardization are essential for reliable interpretation of results across different clinical samples.

How can EPHA10 antibodies be utilized in developing chimeric antigen receptor (CAR) T cell therapy?

EPHA10 antibodies play a critical role in developing effective CAR-T cell therapies through the following methodology:

• Antibody screening and selection: Identification of antibody clones with high specificity for EPHA10 and minimal cross-reactivity with other EphA family members .

• scFv generation: The variable regions of selected anti-EPHA10 antibodies (particularly successful clones like #4) are used to construct single-chain variable fragments (scFvs) that form the antigen-recognition domain of the CAR construct .

• CAR design optimization: Integration of the EPHA10-specific scFv with appropriate costimulatory domains (e.g., CD28, 4-1BB) and CD3ζ signaling domain to create a functional CAR construct.

• Validation studies:

  • In vitro: Assessment of EPHA10 CAR-T cell activity against EPHA10-expressing cancer cells versus control cells

  • In vivo: Evaluation of tumor growth inhibition in relevant preclinical models

Research has demonstrated that EPHA10-specific CAR-T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo, highlighting the therapeutic potential of this approach .

What are the key considerations for studying EPHA10's role in the tumor microenvironment?

Studying EPHA10's role in the tumor microenvironment requires attention to several key considerations:

• Cell type-specific expression analysis:

  • EPHA10 is expressed not only in tumor cells but also in immunosuppressive myeloid cells like TAMs and MDSCs

  • Multiplex immunofluorescence techniques enable visualization of co-localization between EPHA10 and different cell populations

• Functional relevance assessment:

  • Comparing anti-EPHA10 antibody effects in immunocompetent versus immunodeficient models reveals immune-dependent mechanisms

  • Studies have shown that while anti-EPHA10 mAb has significant antitumor effects in immunocompetent BALB/c mice, it shows no antitumor effect in immune-deficient BALB/c-SCID mice

• Mechanism investigation:

  • Examination of changes in tumor-infiltrating lymphocytes (TILs) following anti-EPHA10 treatment

  • Assessment of activation markers on CD8+ T cells (e.g., Granzyme B expression)

  • Quantification of total activated CD8+/GrB+ CTLs and their percentage among CD3+ T cells

• Correlation with other immunosuppressive pathways:

  • EPHA10 expression has been associated with increased PD-L1 expression and immunosuppression

  • Investigating potential synergies between EPHA10-targeted therapies and immune checkpoint inhibitors

These considerations help elucidate how EPHA10 contributes to tumor progression through immune evasion mechanisms and how targeting EPHA10 might restore antitumor immunity.

How can researchers investigate the signaling pathways downstream of EPHA10?

Investigating downstream signaling pathways of EPHA10 requires a multifaceted approach:

Understanding these signaling mechanisms can reveal potential combination therapy strategies that might enhance the efficacy of EPHA10-targeted approaches.

What are the optimal conditions for using biotin-conjugated EPHA10 antibodies in flow cytometry?

For optimal use of biotin-conjugated EPHA10 antibodies in flow cytometry:

• Sample preparation:

  • Fresh samples yield superior results compared to frozen

  • Single-cell suspensions should be prepared with minimal cell death (<10%)

  • FcR blocking step is essential to reduce non-specific binding

• Staining protocol:

  • Titrate the biotin-conjugated EPHA10 antibody to determine optimal concentration (typically 0.1-10 μg/mL)

  • Incubate cells with primary antibody at 4°C for 30-45 minutes

  • Wash thoroughly to remove unbound antibody

  • Detect with fluorophore-conjugated streptavidin (commonly PE, APC, or BV421)

  • Include a secondary staining-only control to assess background

• Controls and validation:

  • Include EPHA10-positive cells (e.g., BT-549 expressing human EPHA10) and matched control cells (mock transfected)

  • Use appropriate isotype control conjugated with biotin

  • Consider comparing results with directly fluorophore-conjugated EPHA10 antibodies if available

• Data analysis considerations:

  • Gate based on viable cells using appropriate viability dye

  • Use fluorescence minus one (FMO) controls to set positive gates accurately

  • Consider density plots rather than histograms for better visualization of positive populations

These optimized conditions maximize signal-to-noise ratio and ensure reliable detection of EPHA10-expressing cells.

How should researchers design experiments to study EPHA10 antibody-mediated internalization?

Studying EPHA10 antibody-mediated internalization requires careful experimental design:

• Antibody selection and labeling:

  • Use biotin-conjugated EPHA10 antibodies or directly labeled fluorescent variants

  • Compare internalization rates between different antibody clones, as they may target different epitopes with varying internalization potentials

• Time-course studies:

  • Establish baseline surface expression at 4°C (prevents internalization)

  • Follow internalization at 37°C at multiple time points (5, 15, 30, 60, 120 minutes)

  • Include controls maintained at 4°C throughout to distinguish internalization from other factors

• Quantification methods:

  • Flow cytometry: Compare surface expression decline over time

  • Confocal microscopy: Track antibody localization from membrane to intracellular compartments

  • pH-sensitive fluorophores: Distinguish surface-bound from internalized antibodies based on pH changes

• Mechanistic investigations:

  • Pharmacological inhibitors of different endocytic pathways (clathrin-mediated, caveolin-mediated, macropinocytosis)

  • Co-localization with endocytic pathway markers (early endosomes, late endosomes, lysosomes)

  • Assessment of antibody degradation versus recycling to the cell surface

• Comparison across cell types:

  • Tumor cells versus immune cells expressing EPHA10

  • Cell lines versus primary tumor samples

Understanding internalization mechanisms is crucial for developing effective antibody-drug conjugates or enhancing therapeutic efficacy of naked antibodies targeting EPHA10.

How can researchers troubleshoot inconsistent results in EPHA10 detection assays?

When facing inconsistent results in EPHA10 detection assays, consider the following troubleshooting approaches:

• Antibody-related factors:

  • Verify antibody specificity through control experiments using EPHA10-positive and negative samples

  • Check for antibody degradation or aggregation (run a small aliquot on SDS-PAGE)

  • Ensure biotin conjugation hasn't compromised the antibody's binding properties

  • Titrate the antibody to identify optimal concentration for specific applications

• Sample preparation issues:

  • Standardize sample collection, processing, and storage procedures

  • For tissue samples, optimize fixation protocols and antigen retrieval methods

  • For flow cytometry, ensure consistent cell dissociation methods that preserve surface epitopes

• Assay-specific considerations:

  • ELISA: Optimize blocking conditions and washing steps to reduce background

  • Flow cytometry: Include viability dye to exclude dead cells that may bind antibodies non-specifically

  • Immunohistochemistry/immunofluorescence: Test multiple antigen retrieval methods

• Expression heterogeneity:

  • EPHA10 expression may vary between cell populations within the same tumor

  • Consider single-cell analysis approaches to address heterogeneity

  • Use multiple markers to identify specific cell populations expressing EPHA10

• Technical validation:

  • Employ multiple detection methods to confirm results

  • Include appropriate positive and negative controls in each experiment

  • Consider using alternative anti-EPHA10 antibody clones recognizing different epitopes

Systematic troubleshooting across these factors can identify the source of inconsistency and lead to more reliable EPHA10 detection protocols.

What are the critical quality control steps for validating biotin-conjugated EPHA10 antibodies?

Critical quality control steps for validating biotin-conjugated EPHA10 antibodies include:

• Conjugation efficiency assessment:

  • Spectrophotometric analysis to determine biotin:protein ratio (optimal range typically 3-8 biotin molecules per antibody)

  • Mass spectrometry to confirm successful conjugation without protein degradation

• Binding capacity verification:

  • Competitive binding assay comparing conjugated versus unconjugated antibody

  • ELISA-based analysis to confirm recognition of recombinant EPHA10 protein

  • Flow cytometry to verify binding to EPHA10-expressing cells (e.g., BT-549 expressing human EPHA10)

• Specificity confirmation:

  • Cross-reactivity testing against other EphA family members (EphA1-EphA8)

  • Binding assessment using EPHA10-knockout or knockdown cells as negative controls

  • Immunoprecipitation followed by Western blotting to confirm target specificity

• Functional activity testing:

  • Ability to induce or block EPHA10 signaling (if applicable)

  • Internalization studies to assess if biotin conjugation affects antibody internalization kinetics

  • If the antibody is intended for therapeutic applications, assess impact on tumor growth inhibition

• Stability evaluation:

  • Accelerated stability studies under different storage conditions

  • Freeze-thaw stability testing

  • Long-term storage assessment with periodic functional testing

These quality control measures ensure that biotin-conjugated EPHA10 antibodies maintain their specificity, sensitivity, and functionality for reliable research applications.

How should researchers interpret changes in EPHA10 expression across different cancer subtypes?

Interpreting changes in EPHA10 expression across cancer subtypes requires consideration of several factors:

These interpretative frameworks help translate EPHA10 expression data into clinically meaningful information for developing targeted therapies across different cancer subtypes.

What are the emerging applications of EPHA10 antibodies beyond cancer research?

While EPHA10 antibodies have been primarily investigated in cancer contexts, several emerging applications expand their utility:

• Diagnostic development:

  • Biomarker detection in liquid biopsies (circulating tumor cells, exosomes)

  • Development of imaging agents for non-invasive tumor detection

  • Multi-parameter diagnostic panels incorporating EPHA10 status

• Therapeutic antibody engineering:

  • Bispecific antibodies targeting EPHA10 and immune effector cells

  • Dimeric EPHA10/CD3 binding bispecific antibodies for anticancer T-cell stimulation

  • Antibody-drug conjugates delivering cytotoxic payloads specifically to EPHA10-expressing cells

• Fundamental biology investigations:

  • Understanding the normal physiological role of EPHA10 in testis

  • Exploring potential functions in embryonic development

  • Investigating evolutionary conservation and divergence of EPHA10 across species

• Reproductive biology applications:

  • Given EPHA10's expression in male testis, exploring its role in spermatogenesis

  • Potential applications in fertility research

• Technology development:

  • As a model system for studying cancer-testis antigens

  • Development of novel antibody formats and conjugation strategies

  • Platform for comparing targeting efficiency of different antibody-based therapeutic modalities

These emerging applications highlight the versatility of EPHA10 antibodies beyond their initial cancer research focus and suggest broader impact in biomedical science.

What are the most promising future directions for EPHA10-targeted therapeutic development?

The most promising future directions for EPHA10-targeted therapeutic development include:

• Enhanced antibody-based therapies:

  • Optimization of antibody clones with improved tumor penetration and effector functions

  • Development of antibody-drug conjugates exploiting the tumor-specific expression of EPHA10

  • Bispecific antibodies co-targeting EPHA10 and immune checkpoint molecules or CD3

• Combination therapy approaches:

  • Integration of EPHA10-targeted therapies with immune checkpoint inhibitors

  • Combination with conventional chemotherapy or radiation therapy

  • Sequential therapy regimens optimized for maximal tumor control

• Advanced CAR-T cell engineering:

  • Building on the promising results of EPHA10-specific CAR-T cells in TNBC models

  • Development of next-generation CAR designs with enhanced persistence and tumor penetration

  • Exploration of allogeneic CAR-T platforms targeting EPHA10

• Broader cancer type applications:

  • Expanding beyond TNBC to other cancer types with EPHA10 expression

  • Stratification approaches to identify patients most likely to benefit from EPHA10-targeted therapies

  • Development of companion diagnostics for patient selection

• Novel therapeutic modalities:

  • RNA-based therapeutics targeting EPHA10 expression

  • Small molecule inhibitors of EPHA10 signaling

  • Peptide-based vaccines eliciting immune responses against EPHA10

These directions leverage the cancer-specific expression pattern of EPHA10 and build upon the promising preclinical results observed with anti-EPHA10 monoclonal antibodies in TNBC models, potentially expanding therapeutic options for patients with limited treatment alternatives.