CLEC14A Antibody

What is CLEC14A and what makes it a promising tumor target?

CLEC14A is a C-type lectin transmembrane protein specifically expressed on the surface of tumor endothelial cells but notably absent in normal tissues under physiological conditions . Its expression is downregulated by shear stress, which explains why it is absent in healthy perfused tissue and only expressed in poorly perfused tissue such as tumors . Studies have shown that CLEC14A is expressed in approximately 30% of human solid tumors, with exceptionally high expression in 50% of human renal tumors . This selective expression pattern makes CLEC14A an attractive target for developing highly specific anti-cancer therapies that would spare normal vasculature, potentially minimizing side effects compared to conventional antiangiogenic approaches.

How can researchers detect and quantify CLEC14A expression in tissue samples?

Immunohistochemistry is the primary method for detecting CLEC14A expression in tissue samples. The recommended protocol involves:

• Preparing serial sections for parallel staining with both CLEC14A antibodies and CD31 (a vascular marker)

• Scoring CLEC14A staining intensity on a scale of 0-4 (0 for no staining and 4 for intense staining)

• Determining the percentage of CLEC14A-positive vessels by comparing the number of CLEC14A-stained vessels to CD31-stained vessels in serial sections

• Calculating the vascular coverage using image analysis software (e.g., ImageJ) to determine the percentage of tissue area occupied by vessels

• Computing the final staining score by multiplying the intensity score, percentage of CLEC14A-positive vessels, and percentage vascular coverage

For mRNA expression analysis, researchers should normalize CLEC14A expression to endothelial markers like PECAM or TIE1. The CLEC14A/TIE1 ratio is particularly effective for distinguishing healthy from tumor tissues, correctly identifying sample status in nearly 75% of cases .

How does CLEC14A function in tumor angiogenesis?

CLEC14A plays a crucial role in tumor angiogenesis through several mechanisms:

• The C-type lectin-like domain (CTLD) of CLEC14A mediates endothelial cell-cell contacts critical for angiogenesis

• CLEC14A is involved in endothelial cell migration and filopodia formation, which are essential processes in forming new blood vessels

• CLEC14A-CTLD promotes VEGF-dependent angiogenesis, making it relevant to multiple cancer types

Functional studies have demonstrated that inhibiting CLEC14A-mediated cell-cell contacts can effectively suppress tumor angiogenesis, highlighting its importance in establishing and maintaining tumor vasculature .

What is the C-type lectin-like domain (CTLD) of CLEC14A and why is it significant?

The C-type lectin-like domain (CTLD) is a key functional domain of CLEC14A that:

• Mediates endothelial cell-cell contacts essential for angiogenesis

• Serves as a specific binding site for therapeutic antibodies

• Is critical for CLEC14A's role in pathological angiogenesis

Research has shown that antibodies specifically targeting CLEC14A-CTLD can directly inhibit CLEC14A-CTLD-mediated endothelial cell-cell contact while simultaneously downregulating expression of CLEC14A on the surface of endothelial cells . This dual mechanism makes the CTLD an ideal target for developing therapeutic antibodies aimed at disrupting tumor angiogenesis.

What mechanisms underlie the efficacy of CLEC14A antibody drug conjugates (ADCs)?

CLEC14A ADCs exert their anti-tumor effects through multiple mechanisms that differ from conventional antiangiogenic approaches:

• Target specificity: Unlike conventional antiangiogenic agents that block tumor neo-angiogenesis, ADCs against CLEC14A disrupt already established tumor vasculature

• Cytotoxic payload delivery: CLEC14A ADCs deliver cytotoxic agents directly to tumor endothelial cells. For example, ADCs linking anti-CLEC14A antibodies (such as CRT3 and CRT4) to SG3249, a pyrrolobenzodiazepine dimer-based DNA crosslinking agent, have shown significant efficacy

• Dual action mechanism: These ADCs demonstrate both:

  • Direct cytotoxicity against endothelial cells in vitro

  • Reduced tumor vascularization in vivo, correlating with reduced tumor burden

• Targeted vascular damage: In animal models, CLEC14A ADCs cause leakage of blood into surrounding tissues within 24 hours of administration, indicating selective vascular damage within the tumor microenvironment while sparing normal tissues

In murine models of Lewis lung carcinoma, two weekly treatments with C4-SG3249 significantly reduced tumor weight compared to control antibodies, with no adverse effects observed in healthy tissues following ADC administration .

How do researchers optimize antibodies targeting CLEC14A-CTLD?

Optimizing antibodies targeting CLEC14A-CTLD involves several sophisticated approaches:

• Complementarity-determining region (CDR) grafting: This technique transfers the antigen-binding regions from one antibody to another to improve specificity and function

• Consecutive deglycosylation: Removing specific glycosylation sites can enhance stability and homogeneity of the antibody

• Functional isolation: Screening antibody variants based on their functional properties to select those with optimal anti-angiogenic effects

• Specificity validation: Using methodologies such as:

  • Transducing cells with CLEC14A genes and co-expressing GFP

  • Flow cytometric analysis with titrating concentrations of candidate antibodies

  • Comparing staining between transduced cells and control vector cells

These optimization approaches have yielded novel anti-angiogenic human monoclonal antibodies with improved stability and homogeneity compared to parental antibodies, as demonstrated with antibodies effectively suppressing VEGF-dependent angiogenesis and tumor angiogenesis across multiple cancer models .

What experimental models have been validated for studying CLEC14A antibodies?

Several experimental models have been validated for studying CLEC14A antibodies:

• In vitro models:

  • Human umbilical vascular endothelial cells (HUVECs) for cytotoxicity assays

  • Endothelial cell migration and tube formation assays to assess angiogenic inhibition

  • Cell-cell contact assays to evaluate CLEC14A-CTLD interactions

• In vivo models:

  • Lewis lung carcinoma in C57BL6 mice for assessing vascular damage and tumor burden reduction

  • Xenograft models using:

    • SNU182 human hepatocellular carcinoma

    • CFPAC-1 human pancreatic cancer

    • U87 human glioma cells

    • HCT116 human colorectal cancer, including bevacizumab-adapted variants

• Biodistribution studies:

  • Intravenous administration of antibodies (CRT3, CRT4) in tumor-bearing mice with analysis of tissue distribution after 90 minutes

  • Immunostaining to visualize antibody localization in tumors and healthy tissues

These models consistently demonstrate that CLEC14A antibodies predominantly localize to tumor vessels with minimal binding to healthy tissues, confirming the tumor-specificity of this target .

How does CLEC14A antibody therapy compare to VEGF-dependent angiogenesis inhibitors?

CLEC14A antibody therapy offers several distinct advantages over traditional VEGF-dependent angiogenesis inhibitors:

• Complementary targeting mechanism: While VEGF inhibitors block new vessel formation, CLEC14A antibodies disrupt already established tumor vasculature, suggesting potential for combination approaches

• Efficacy in bevacizumab-resistant tumors: Studies have shown that optimized antibodies targeting CLEC14A-CTLD significantly inhibit tumor angiogenesis in bevacizumab-adapted HCT116 human colorectal cancer cells, suggesting activity in tumors that have developed resistance to VEGF inhibition

• Tumor specificity: CLEC14A's highly selective expression in tumor endothelial cells but not normal vasculature potentially offers improved safety profile compared to VEGF inhibitors, which target factors present in both tumor and normal angiogenesis

• Dual mechanism of action: Anti-CLEC14A antibodies not only block CLEC14A-CTLD-mediated endothelial cell-cell contact but also downregulate CLEC14A expression on endothelial cell surfaces, providing a multi-faceted approach to angiogenesis inhibition

These properties suggest that CLEC14A antibody therapy may address some limitations of current VEGF-targeted therapies while potentially offering new options for patients with VEGF-resistant tumors.

What methodologies are most effective for validating CLEC14A antibody specificity?

Ensuring antibody specificity is critical for CLEC14A research. The following methodologies have proven effective:

• Comparative immunohistochemistry:

  • Staining serial sections with both CLEC14A antibodies and vascular markers like CD31

  • Using concentration-matched isotype control antibodies as negative controls

  • Comparing staining patterns across multiple tissue types (tumor vs. healthy)

• Cross-species validation:

  • Testing antibody binding across human and non-human primate tissues

  • Comparing staining patterns between species to confirm conserved epitope recognition

• Cell line validation:

  • Transducing 293T cells with CLEC14A genes and co-expressing GFP as a marker

  • Flow cytometric analysis with titrating concentrations of the test antibody

  • Comparing staining between CLEC14A-expressing and control vector cells

• Functional assays:

  • Assessing the antibody's ability to block known CLEC14A functions (cell migration, tube formation)

  • Confirming specific inhibition of CLEC14A-mediated cell-cell contacts

  • Verifying that antibody treatment downregulates CLEC14A expression on endothelial cell surfaces

Using multiple validation approaches increases confidence in antibody specificity, which is crucial for both research applications and potential therapeutic development.

What analytical approaches help resolve contradictions in CLEC14A expression data?

Researchers may encounter contradictory data regarding CLEC14A expression across different studies. The following analytical approaches help resolve such discrepancies:

These approaches have helped researchers establish that CLEC14A mRNA expression per endothelial cell is significantly higher in tumors compared to healthy tissues, with the CLEC14A/TIE1 ratio alone allowing correct identification of sample status in approximately 75% of cases .