AXL1 Antibody

What is AXL receptor tyrosine kinase and what is its significance in cancer research?

AXL receptor tyrosine kinase belongs to the TAM (Tyro3/Axl/MerTK) family of receptor tyrosine kinases. It functions as a negative regulator of innate immunity and plays critical roles in multiple cellular processes. In cancer biology, AXL has significant relevance as its activation through its ligand Gas6 drives tumor growth, metastasis, and is associated with acquired resistance to targeted therapies, radiotherapy, and chemotherapy . AXL protein expression has been demonstrated in 55-70% of pancreatic cancers and is associated with aggressive tumor behavior, higher frequency of distant metastases, and poor survival . Structurally, the protein has a molecular weight of approximately 98.3 kilodaltons, though in Western blotting applications it often appears as a band of approximately 138 kDa in cell lysates due to post-translational modifications .

How can researchers differentiate between phosphorylated and total AXL in experimental settings?

Researchers should employ specific antibodies that recognize either the phosphorylated form (p-AXL) or total AXL protein. When conducting studies examining AXL activation, it's crucial to analyze both forms simultaneously:

• For phosphorylated AXL detection:

  • Use phospho-specific antibodies that recognize specific phosphorylation sites

  • Include positive controls such as Gas6-stimulated cells to validate phosphorylation state

  • Consider phosphatase inhibitors in lysate preparation

• For total AXL detection:

  • Use antibodies recognizing domains independent of phosphorylation status

  • Western blotting typically shows AXL as a band of approximately 138 kDa

When analyzing both forms, use quantitative analysis to determine the ratio of phosphorylated to total AXL as a measure of activation. Studies have demonstrated correlation between p-AXL and total AXL expression levels in various cancer specimens, particularly in triple-negative breast cancer where they positively correlate with KLF5 expression .

What sample preparation techniques optimize AXL detection in different experimental systems?

Optimal sample preparation for AXL detection varies by experimental approach:

For Western blotting:

• Use 25-40 μg of total cell lysate for optimal AXL and phospho-AXL detection

• Include protease and phosphatase inhibitors in lysis buffers

• Employ denaturating conditions with proper reducing agents

• For membrane proteins like AXL, consider detergent selection carefully

For immunohistochemistry:

• Appropriate fixation is critical; formalin-fixed paraffin-embedded tissues have been successfully used to detect AXL expression patterns in tumor specimens

• Antigen retrieval steps are essential for exposing the epitope

• Signal amplification may be necessary for detecting lower expression levels

• Optimal antibody dilution should be determined empirically for each application

In clinical specimens, validation with multiple antibodies may be necessary to confirm specificity of staining patterns, as demonstrated in studies of pancreatic adenocarcinoma where AXL was detected in 76% of cases with stronger staining observed in invasive cells .

What are the validated applications for anti-AXL antibodies in experimental research?

Anti-AXL antibodies have been validated for multiple experimental applications, each requiring specific optimization:

• Western Blotting:

  • Detects AXL at approximately 138 kDa in human cell lysates

  • Can distinguish between phosphorylated and total protein forms

  • Requires optimization of antibody concentration, blocking conditions, and incubation times

• Immunohistochemistry:

  • Enables visualization of AXL expression patterns in tissue samples

  • Has been used to demonstrate AXL expression in 76% of pancreatic ductal adenocarcinoma cases

  • Allows for analysis of spatial distribution, showing stronger AXL staining in invasive cells at tumor periphery

• Immunofluorescence:

  • Enables subcellular localization studies

  • Used to demonstrate internalization of anti-AXL antibodies into cancer cells

• Flow Cytometry:

  • Quantifies AXL expression levels on cell surfaces

  • Useful for sorting AXL-positive cellular populations

• Immunoprecipitation:

  • Isolates AXL protein complexes for downstream analysis

  • Enables study of AXL-associated proteins and signaling complexes

The choice of application should be guided by the specific research question, with appropriate validation controls included in experimental design.

How can researchers effectively validate the specificity of anti-AXL antibodies?

Validation of anti-AXL antibody specificity is critical for reliable experimental results and should include:

• Molecular weight verification:

  • Confirm detection of a protein at the expected molecular weight (approximately 138 kDa for AXL in Western blotting)

  • Check for absence of non-specific bands

• Positive and negative controls:

  • Use cell lines with known AXL expression levels as positive controls

  • Include AXL-negative cell lines or AXL-knockdown samples as negative controls

  • Studies have successfully used shRNA-mediated knockdown of AXL in Panc-1 cells to demonstrate antibody specificity

• Peptide competition assays:

  • Pre-incubate antibody with purified AXL protein or peptide

  • Observe elimination of specific signal

• Cross-validation with multiple antibodies:

  • Use different antibodies targeting distinct epitopes of AXL

  • Compare staining/detection patterns for consistency

• Functional validation:

  • Confirm antibody effects on known AXL functions (e.g., inhibition of Gas6 binding, blocking of AXL phosphorylation)

  • Evidence from studies shows effective antibodies inhibit Gas6-dependent AXL phosphorylation and downstream signaling

Proper validation increases confidence in experimental results and prevents misinterpretation of data due to non-specific antibody binding.

What controls are essential when using anti-AXL antibodies for signaling pathway analysis?

When investigating AXL-mediated signaling pathways, essential controls include:

• Stimulation controls:

  • Compare unstimulated vs. Gas6-stimulated samples to confirm pathway activation

  • Include time-course analysis to capture optimal activation windows

• Inhibition controls:

  • Use known AXL inhibitors (e.g., specific antibodies or small molecule inhibitors) to confirm signal specificity

  • Anti-AXL mAbs like D9 and E8 have been shown to inhibit phosphorylation of AXL and its downstream target AKT

• Knockdown/knockout validation:

  • Include AXL-knockdown or knockout samples to validate pathway-specific effects

  • Studies have demonstrated that AXL-knockdown Panc-1 cells showed decreased migration, survival, proliferation, and reduced tumor growth in vivo

• Downstream marker analysis:

  • Monitor known AXL effectors (e.g., AKT, ERK) to confirm pathway engagement

  • Phosphorylation status of AKT has been established as a reliable indicator of AXL signaling

• Cross-pathway controls:

  • Include markers for related pathways to assess specificity and cross-talk

  • Consider potential redundancy with other TAM family receptors (Tyro3, MerTK)

Using these controls ensures that observed effects are specifically related to AXL signaling and not due to off-target effects or experimental artifacts.

How do anti-AXL antibodies affect the epithelial-mesenchymal transition (EMT) in cancer models?

Anti-AXL antibodies have demonstrated significant effects on epithelial-mesenchymal transition (EMT), a process strongly linked to AXL activity:

• Mechanism of action:

  • AXL has been demonstrated to be required for EMT of malignant cells induced by various stimuli, including H-RASV12 and overexpression of SLUG

  • Anti-AXL antibodies can interfere with this process by:
    a) Blocking Gas6-mediated AXL activation
    b) Inducing AXL internalization and downregulation
    c) Inhibiting downstream signaling pathways essential for EMT maintenance

• Experimental evidence:

  • Studies have shown that anti-AXL mAbs (D9 and E8) induce down-expression of AXL by internalization within 1.5 hours of exposure

  • This internalization leads to inhibition of AXL phosphorylation by Gas6, disrupting EMT-supporting signaling

  • In pancreatic cancer cell lines, anti-AXL antibodies reduced cell migration, a key EMT-associated phenotype

• Impact on EMT markers:

  • Anti-AXL antibody treatment can restore epithelial marker expression (E-cadherin)

  • Treatment reduces mesenchymal markers (N-cadherin, vimentin)

  • Affects EMT-inducing transcription factors (Snail, Slug, ZEB1)

• Relevance to cancer progression:

  • Cancers that have undergone EMT display increased invasiveness, metastatic capacity, and multidrug resistance

  • In pancreatic cancer tissues, stronger AXL staining was observed in invasive cells at the tumor periphery and in emboli, supporting its role in invasion and dissemination

Understanding these mechanisms allows researchers to effectively utilize anti-AXL antibodies to study and potentially target EMT in cancer progression.

How can anti-AXL antibodies be used to investigate therapy resistance mechanisms?

Anti-AXL antibodies serve as valuable tools for investigating therapy resistance mechanisms across multiple cancer types:

• AXL overexpression in resistant populations:

  • AXL overexpression is associated with resistance to standard chemotherapy and tyrosine kinase inhibitors in multiple cancers

  • Documented resistance mechanisms include:
    a) Acute and chronic myeloid leukemia resistance to standard therapy
    b) Gastrointestinal stromal tumor resistance to TKIs
    c) Breast, ovarian, and lung cancer therapy resistance

• Methodological approaches:

  • Use anti-AXL antibodies to screen resistant vs. sensitive cell populations

  • Compare AXL expression levels before and after exposure to therapeutic agents

  • Monitor AXL phosphorylation status during resistance development

  • Example: ERL-resistant lung cancer cell lines showed increased AXL expression and phosphorylation compared to parental cell lines

• Intervention studies:

  • Anti-AXL antibodies can be used to determine if AXL inhibition resensitizes resistant cells

  • Combined blockade approaches (e.g., AXL with PD-L1) show promise for overcoming resistance

  • A humanized IgG1 Axl-targeting monoclonal antibody (CDX-0168) inhibits Gas6-dependent Axl phosphorylation and signaling, potentially addressing resistance mechanisms

• Downstream signaling analysis:

  • Anti-AXL antibodies enable tracking of altered signaling in resistant cells

  • Common pathways involved include PI3K/AKT and MAPK/ERK cascades

By systematically applying these approaches, researchers can elucidate the specific mechanisms by which AXL contributes to therapy resistance and develop strategies to overcome them.

What immunological effects do anti-AXL antibodies elicit in the tumor microenvironment?

Anti-AXL antibodies demonstrate significant immunomodulatory effects in the tumor microenvironment through multiple mechanisms:

• AXL's role in immune suppression:

  • AXL functions as a negative regulator of innate immunity

  • Activation of AXL through Gas6 leads to suppression of myeloid cell activity, promoting an immunosuppressive tumor microenvironment

• Antibody-mediated immune activation:

  • Humanized IgG1 anti-AXL antibodies (e.g., CDX-0168) induce potent release of pro-inflammatory cytokines and chemokines from:
    a) Dendritic cells
    b) Monocytes
    c) Macrophages

  • This effect occurs through an Fc receptor-dependent mechanism

  • Treatment enhances T cell activation in mixed lymphocyte reactions

• Combination strategies:

  • Anti-AXL antibodies may enhance antitumor activity associated with PD-(L)1 blockade

  • Bispecific antibodies targeting both AXL and PD-L1 (e.g., tetravalent bispecific Axl x PD-L1 antibody) show greater cytokine release and T cell activation than combinations of individual antibodies

• Antibody-dependent cellular cytotoxicity (ADCC):

  • Anti-AXL antibodies like CDX-0168 elicit tumor cell killing via ADCC both in vitro and in vivo

  • This provides an additional mechanism of anti-tumor activity beyond signaling inhibition

These immunomodulatory effects represent an important dimension of anti-AXL antibody function, supporting their potential application in cancer immunotherapy approaches.

What are the critical parameters for successful Western blotting with anti-AXL antibodies?

Successful Western blotting with anti-AXL antibodies requires attention to several critical parameters:

• Sample preparation:

  • Use adequate protein amounts (25-40 μg of total cell lysate is recommended for AXL detection)

  • Include both phosphatase and protease inhibitors in lysis buffers

  • Ensure complete solubilization of membrane proteins like AXL

  • HeLa cell lysates have been validated as positive controls for AXL detection

• Gel electrophoresis considerations:

  • Use appropriate percentage gels (typically 7.5-10%) for optimal resolution of AXL (138 kDa)

  • Consider gradient gels for better separation of high molecular weight proteins

  • Allow sufficient run time for proper resolution of high molecular weight bands

• Transfer optimization:

  • Use wet transfer methods for large proteins like AXL

  • Extend transfer times or reduce current for better transfer efficiency

  • Consider transfer buffers optimized for high molecular weight proteins

• Antibody selection and dilution:

  • Choose validated antibodies with confirmed specificity for AXL

  • PrecisionAb monoclonal antibodies like clone 7E10 have been validated for detection of AXL bands at approximately 138 kDa in human cell lysates

  • Determine optimal antibody concentration through titration experiments

• Common issues and solutions:

  • Weak signals: Increase protein loading, optimize antibody concentration, extend exposure times

  • Multiple bands: Validate specificity using knockdown controls, test different antibody clones

  • Background issues: Increase blocking time, optimize washing steps, reduce antibody concentration

Following these guidelines will help ensure reproducible and reliable detection of AXL in Western blotting applications.

What strategies can optimize immunohistochemical detection of AXL in tissue specimens?

Optimizing immunohistochemical detection of AXL in tissue specimens requires attention to several key parameters:

• Tissue preparation and fixation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues have been successfully used for AXL detection

  • Consistent fixation times are critical for reproducible results

  • Consider tissue-specific optimization of fixation protocols

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) is typically necessary for AXL detection

  • Compare citrate-based (pH 6.0) versus EDTA-based (pH 9.0) retrieval buffers

  • Optimize retrieval times and temperatures for specific tissue types

• Antibody selection and validation:

  • Test multiple anti-AXL antibodies targeting different epitopes

  • Validate specificity using positive and negative control tissues

  • Determine optimal antibody dilutions through titration experiments

• Signal development considerations:

  • Compare DAB versus other chromogens for optimal visualization

  • Consider signal amplification methods for low-expression samples

  • Implement standardized development times for consistency across specimens

• Scoring and evaluation methods:

  • Define clear criteria for positive staining (membrane, cytoplasmic, or both)

  • Consider intensity scoring scales (e.g., mild positive (+), moderate positive (++), strong positive (+++)) as used in previous studies

  • Evaluate specific patterns such as stronger staining in invasive cells at tumor periphery

Studies have successfully used these approaches to demonstrate AXL expression in 76% of pancreatic ductal adenocarcinoma cases, with 66% of AXL-positive tumors showing stronger staining in invasive cells .

How can researchers effectively troubleshoot antibody internalization experiments with anti-AXL antibodies?

Antibody internalization experiments are crucial for understanding the mechanisms of anti-AXL antibody action. Effective troubleshooting requires:

• Temperature control protocols:

  • Compare internalization at 37°C (active internalization) versus 4°C (surface localization only)

  • Previous studies have shown that anti-AXL mAbs (D9 and E8) are internalized in cancer cells following incubation at 37°C for 1 hour but remain localized at the cell surface after incubation at 4°C

  • Maintain strict temperature control throughout experiments

• Time-course optimization:

  • Design experiments with multiple time points (e.g., 0.5, 1, 1.5, 3, 6 hours)

  • Research has shown that AXL down-regulation begins as early as 1.5 hours after exposure to anti-AXL antibodies

  • Use consistent timing protocols across experimental replicates

• Visualization techniques:

  • Immunofluorescence microscopy can effectively track antibody localization

  • Consider:
    a) Direct fluorophore conjugation to anti-AXL antibodies
    b) Secondary antibody detection systems
    c) Co-localization with endosomal/lysosomal markers

• Quantification approaches:

  • Flow cytometry to measure surface versus internalized antibody levels

  • Image analysis software for quantifying intracellular fluorescence

  • Western blotting to assess total AXL protein levels after internalization

• Common challenges and solutions:

  • Poor internalization: Confirm antibody binding to correct epitope, verify cell viability

  • High background: Optimize washing procedures, adjust antibody concentrations

  • Cell type variation: Different cell lines may show varying internalization kinetics

Proper implementation of these approaches allows researchers to accurately characterize the internalization dynamics of anti-AXL antibodies, which is critical for understanding their mechanisms of action in downregulating AXL signaling.

How are bispecific antibodies targeting AXL revolutionizing cancer immunotherapy approaches?

Bispecific antibodies targeting AXL represent an innovative frontier in cancer immunotherapy research:

• Mechanism and design considerations:

  • Tetravalent bispecific AXL x PD-L1 antibodies combine anti-AXL with anti-PD-L1 mAbs in an IgG-scFv format

  • This approach simultaneously targets:
    a) AXL-mediated tumor growth and metastasis
    b) PD-L1-mediated immune checkpoint blockade

  • CDX-0168 (anti-AXL) combined with 9H9 (anti-PD-L1) has demonstrated promising results in preclinical studies

• Enhanced immune activation:

  • Bispecific antibodies elicit greater cytokine release and T cell activation in vitro than combinations of parental antibodies

  • They maintain robust dual pathway blockade of both AXL and PD-L1 signaling

• Advantages over combination therapy:

  • Simplified administration of a single agent

  • Potential synergistic effects from co-targeting within the same molecule

  • Consistent ratio of targeting moieties

• Current research status:

  • Preclinical development is demonstrating promising results

  • Additional studies investigating simultaneous blockade of AXL and PD-L1 pathways with various agents are underway

  • This approach represents a novel anti-cancer therapeutic strategy with significant potential

The continued development of bispecific antibodies targeting AXL in combination with immune checkpoint inhibitors could potentially address multiple cancer hallmarks simultaneously, improving therapeutic outcomes.

What methodological approaches best assess the impact of anti-AXL antibodies on cancer stem cell populations?

Assessing the impact of anti-AXL antibodies on cancer stem cell (CSC) populations requires specialized methodological approaches:

• In vitro analysis techniques:

  • Sphere formation assays to measure self-renewal capacity

  • Flow cytometry for CSC marker expression (CD44, CD133, ALDH activity)

  • Serial limiting dilution assays to quantify stem cell frequency

  • Gene expression analysis of stemness-associated pathways

• In vivo tumorigenicity assays:

  • Limited dilution transplantation studies after antibody treatment

  • Research has demonstrated that AXL inhibition (using DCC-2036) affected CSC frequency in 4T1 cells as determined by in vivo tumorigenicity assays

  • Transplantation of varying cell numbers (e.g., 200,000, 20,000, or 2,000 cells) to calculate CSC frequency using ELDA (Extreme Limiting Dilution Analysis)

• Molecular mechanism investigation:

  • Analysis of AXL co-expression with stemness factors (e.g., KLF5)

  • Immunohistochemical analysis of tumor tissues for stemness markers

  • Western blotting to assess changes in stemness-related signaling pathways

• Resistance and recurrence models:

  • Development of resistant cell populations after serial antibody treatment

  • Analysis of AXL expression in tumor recurrence models

  • Comparison of primary and metastatic lesions for AXL and CSC marker expression

These methodological approaches provide comprehensive assessment of how anti-AXL antibodies affect cancer stem cell populations, potentially addressing a key mechanism of therapy resistance and tumor recurrence.

How can researchers effectively combine anti-AXL antibodies with other targeted therapies in experimental models?

Effective combination of anti-AXL antibodies with other targeted therapies requires systematic experimental design:

• Rational selection of combination partners:

  • AXL upregulation occurs in resistance to various therapies including:
    a) Tyrosine kinase inhibitors in myeloid leukemia and gastrointestinal stromal tumors
    b) Conventional chemotherapy in breast, ovarian, and lung cancers

  • Select complementary targets based on:
    a) Pathway analysis (e.g., combining AXL inhibition with downstream effector blockade)
    b) Resistance mechanisms (e.g., combining with the primary therapy causing AXL-mediated resistance)
    c) Immune modulatory potential (e.g., combining with immune checkpoint inhibitors)

• Synergy assessment methodologies:

  • Dose-effect curves calculated using computational tools (e.g., CompuSyn software)

  • For combination treatments, plot combined drug doses and analyze for synergistic, additive, or antagonistic effects

  • Implement robust statistical analysis for interaction assessment

• Sequence-dependent effects:

  • Compare different treatment sequences:
    a) Concurrent administration
    b) Sequential administration (anti-AXL antibody first followed by partner therapy)
    c) Alternating schedules

  • Document temporal dynamics of pathway inhibition

• Multidimensional endpoint analysis:

  • Assess combination effects on:
    a) Cell proliferation and survival
    b) Migration and invasion capabilities
    c) Cancer stem cell properties
    d) Immune system activation
    e) In vivo tumor growth and metastasis formation

• Translation to in vivo models:

  • Use both subcutaneous and orthotopic xenograft models

  • Consider syngeneic models for immune-related combinations

  • Implement patient-derived xenografts for clinical relevance

The systematic implementation of these approaches enables researchers to identify and characterize the most promising combination strategies involving anti-AXL antibodies, potentially leading to more effective therapeutic interventions.