CLDN4 Antibody

What is CLDN4 and why is it significant in cancer research?

CLDN4 (Claudin 4) is a 22.1 kDa membrane protein consisting of 209 amino acids that integrates into tight junctions to form molecular barriers at paracellular spaces between endothelial and epithelial cells . It serves as the backbone of tight junction structure and function, making it an attractive target for modulating tissue permeability to deliver drugs or treat disease . CLDN4 is highly expressed in multiple tumor types including non-small cell lung cancer (NSCLC), urothelial carcinoma (UC), colorectal cancer (CRC), prostate adenocarcinoma (PA), ovarian cancer (OC), and triple-negative breast cancer (TNBC) . Recent studies have demonstrated its particular significance in ovarian cancer, where it is substantially upregulated compared to normal tissue samples .

What detection methods are most effective for CLDN4 expression analysis?

For effective CLDN4 expression analysis, multiple complementary techniques should be employed:

• Western Blot (WB): Effective for quantifying CLDN4 protein levels and validating antibody specificity. Recommended for initial characterization studies.

• Immunohistochemistry (IHC): Critical for visualizing CLDN4 expression patterns in tissue sections and determining localization. Studies have effectively used IHC to demonstrate significantly higher CLDN4 expression in ovarian cancer samples compared to nontumor tissues .

• Flow Cytometry (FCM): Valuable for quantifying CLDN4 expression in cell populations and for sorting CLDN4-positive cells.

• RT-PCR: Essential for mRNA expression analysis, which has been successfully used to confirm elevated CLDN4 expression in ovarian cancer specimens compared to nontumor tissues .

• ROC (Receiver Operating Characteristic) Analysis: Statistical technique used to evaluate diagnostic potential. ROC testing has revealed that CLDN4 effectively distinguishes ovarian cancer specimens from normal specimens with an area under the ROC curves (AUC) of 0.993 (95% confidence interval: 0.983 to 1.000) .

How do I validate the specificity of a CLDN4 antibody?

Validating CLDN4 antibody specificity requires a multi-faceted approach:

• Positive and Negative Controls: Include tissues/cell lines known to express high levels of CLDN4 (e.g., ovarian cancer samples) alongside negative controls lacking CLDN4 expression.

• Cross-Reactivity Testing: Test antibody against related claudin family members, particularly those with high sequence homology, to ensure specificity. This is especially important as CLDN4 may share structural similarities with other claudin proteins.

• siRNA Knockdown Validation: Perform CLDN4 knockdown experiments using siRNA in positive control cells, then confirm reduced antibody binding.

• Western Blot Analysis: Verify that the antibody detects a single band at the expected molecular weight of approximately 22.1 kDa .

• Peptide Competition Assay: Pre-incubate antibody with purified CLDN4 protein or peptide to block specific binding sites, which should diminish or eliminate target detection in positive samples.

What evidence supports CLDN4 as a diagnostic biomarker in ovarian cancer?

Multiple lines of evidence support CLDN4's utility as a diagnostic biomarker in ovarian cancer:

• Expression Analysis: CLDN4 is significantly upregulated in ovarian cancer samples compared to nontumor tissues, as demonstrated in both TCGA and GSE26712 datasets .

• ROC Analysis Performance: CLDN4 discriminates ovarian cancer from normal tissue with remarkable accuracy, showing an AUC of 0.993 (95% CI: 0.983 to 1.000) . This exceptionally high value indicates excellent diagnostic potential.

• Validation in Independent Cohorts: The diagnostic value has been confirmed across multiple independent patient cohorts, strengthening its reliability as a biomarker .

• Multi-Cancer Profiling: Pancancer investigations revealed dysregulated CLDN4 expression across multiple tumor types, suggesting its broader relevance in cancer diagnostics .

• RT-PCR Validation: Independent RT-PCR experiments have confirmed significantly elevated CLDN4 expression in ovarian cancer specimens compared to nontumor tissues, validating findings from larger datasets .

How does CLDN4 expression correlate with prognostic outcomes in cancer patients?

CLDN4 expression demonstrates significant prognostic value, particularly in ovarian cancer:

What is the relationship between CLDN4 expression and immune cell infiltration in tumors?

CLDN4 expression shows significant associations with immune cell infiltration in tumor microenvironments:

• Positive Correlations: CLDN4 expression is positively associated with infiltration of specific immune cell types, including:

  • Th17 cells (T helper 17 cells)

  • NK CD56bright cells (Natural Killer cells)

• Negative Correlations: CLDN4 expression is negatively associated with:

  • Th2 cells (T helper 2 cells)

  • pDC (plasmacytoid dendritic cells)

  • T helper cells

• Macrophage Dynamics: M2 macrophages play a particularly significant role in the tumor microenvironment related to CLDN4 expression. These macrophages develop from macrophages in the complex tumor microenvironment and significantly regulate tumor growth, invasion, and metastasis .

• Immunotherapeutic Implications: These correlations suggest CLDN4 may influence immunotherapeutic responses and could potentially serve as a marker for immunotherapy selection.

What are the optimal techniques for developing claudin-targeting antibodies?

Developing effective claudin-targeting antibodies presents unique challenges due to claudins' small size and complex physicochemical properties. Optimal techniques include:

• Synthetic Antibody Fragment (sFab) Technology: Recent research has successfully employed sFab-encoding phage display libraries against detergent-solubilized human CLDN-4 bound to cCpE (C-terminal fragment of Clostridium perfringens enterotoxin) . This approach has yielded high-affinity binders termed CpE Obstructing Proteins (COPs).

• Structural-Guided Design: Utilizing high-resolution structures (2.2–2.6 Å) of CLDN-4/cCpE complexes determined by cryo-EM to inform antibody design strategies. This approach exceeds the resolution achieved previously with X-ray crystallography .

• Bispecific Antibody Development: Creating bispecific antibodies that simultaneously target CLDN4 and immune modulators (e.g., CD137) represents an innovative approach to enhance antitumor immunity, as demonstrated by ASP1002 .

• Selection Against Native Conformations: Ensuring antibodies recognize native conformations of CLDN4 as it exists in tight junctions, rather than just denatured protein fragments.

• Claudin-Selectivity Screening: Implementing rigorous screening to identify antibodies with selectivity for CLDN4 over other homologous claudins, as achieved with COP-1 which demonstrates CLDN4-selective binding .

How can researchers optimize CLDN4 antibody staining protocols for various tissue types?

Optimizing CLDN4 antibody staining requires tissue-specific adjustments:

• Antigen Retrieval Optimization:

  • Epithelial tissues: Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Ovarian tissues: EDTA buffer (pH 9.0) has shown superior results for CLDN4 detection

  • Fixed tissues: Proteinase K digestion may be necessary to expose CLDN4 epitopes

• Blocking Protocol Enhancement:

  • Include both protein blocking (5% BSA or serum) and permeabilization steps

  • For tissues with high endogenous biotin, employ avidin/biotin blocking system

  • Consider dual blocking with normal serum plus 0.3% Triton X-100 for improved specificity

• Signal Amplification Strategies:

  • Employ tyramide signal amplification for formalin-fixed paraffin-embedded (FFPE) tissues

  • Consider fluorescent secondary antibodies for co-localization studies

  • Use polymer-based detection systems for IHC of clinical samples

• Background Reduction Techniques:

  • Include 0.1-0.3% Triton X-100 in wash buffers

  • Extend blocking time to 2 hours at room temperature

  • Use 0.1% Tween-20 in all wash steps

• Validation Controls:

  • Always include positive control tissues with known CLDN4 expression

  • Include antibody omission controls and isotype controls

  • Consider nuclear counterstaining with DAPI or hematoxylin to improve localization assessment

What statistical methods are most appropriate for analyzing CLDN4 expression data in clinical samples?

For robust statistical analysis of CLDN4 expression data in clinical samples:

• For Paired Sample Comparisons:

  • Wilcoxon signed-rank test is recommended for analyzing paired samples (e.g., tumor vs. adjacent normal tissue from the same patient)

• For Unpaired Sample Comparisons:

  • Wilcoxon rank-sum test should be employed for unpaired samples (e.g., comparing CLDN4 expression between patient groups)

• For Diagnostic Performance Assessment:

  • Receiver Operating Characteristic (ROC) curve analysis with area under the curve (AUC) calculation

  • Calculate sensitivity, specificity, positive predictive value, and negative predictive value at optimal cutoff points

• For Correlations with Clinical Features:

  • Chi-square test or Fisher's exact test for categorical variables (e.g., correlating CLDN4 expression with clinical stage or grade)

  • A p-value threshold of 0.05 should be used to determine statistical significance

• For Survival Analysis:

  • Kaplan-Meier analysis with log-rank test to compare survival outcomes between high and low CLDN4 expression groups

  • Cox proportional hazards regression for multivariate analysis, adjusting for confounding factors

How is CLDN4 being targeted in current therapeutic development?

CLDN4 is being targeted through multiple innovative therapeutic approaches:

• Bispecific Antibody Development: ASP1002 represents a cutting-edge approach as a bispecific antibody designed to simultaneously target CLDN4 and CD137. This dual targeting enhances antitumor T cell responses against CLDN4-expressing tumor cells . A phase 1 first-in-human, open-label, multicenter study (NCT05719558) is currently evaluating its safety, tolerability, and efficacy .

• Tight Junction Modulation: Researchers are developing antibodies that can specifically modulate tight junction permeability by targeting CLDN4, potentially enhancing drug delivery across biological barriers .

• Synthetic Antibody Fragments: The development of synthetic antibody fragments (sFabs) that bind human CLDN4 with high specificity represents a novel approach for both imaging and therapeutic applications .

• Antibody-Drug Conjugates: While not explicitly mentioned in the search results, the selective expression of CLDN4 in multiple cancer types makes it an attractive target for antibody-drug conjugate development.

• Combinatorial Approaches: Integrating CLDN4-targeting with immune checkpoint inhibitors or conventional chemotherapies to enhance therapeutic efficacy.

What challenges must be overcome in developing CLDN4-targeted therapeutics?

Several significant challenges exist in developing effective CLDN4-targeted therapeutics:

• Structural Complexity: Claudins' small size (22 kDa for CLDN4) and complex physicochemical properties make them difficult targets for traditional antibody development approaches .

• Specificity Concerns: Ensuring sufficient selectivity for CLDN4 over other claudin family members represents a major challenge given their structural similarities.

• Accessibility Issues: CLDN4's localization within tight junctions may limit accessibility for therapeutic antibodies, requiring specialized delivery strategies.

• Safety Considerations: Since CLDN4 plays physiological roles in normal tissues, potential off-target effects must be carefully evaluated during therapeutic development.

• Resistance Mechanisms: Potential development of resistance through altered CLDN4 expression, localization, or compensatory upregulation of other claudin family members needs to be investigated.

How do CLDN4 antibodies compare in sensitivity and specificity across different applications?

CLDN4 antibodies demonstrate variable performance characteristics across applications:

• Western Blotting Applications:

  • Monoclonal antibodies typically show greater specificity but sometimes reduced sensitivity

  • Polyclonal antibodies often provide increased sensitivity but may show cross-reactivity with other claudins

  • Recombinant antibodies demonstrate consistent batch-to-batch performance with good specificity

• Immunohistochemistry Performance:

  • Rabbit polyclonal antibodies generally show superior sensitivity for FFPE tissues

  • Mouse monoclonals may require more rigorous antigen retrieval but often demonstrate better specificity

  • Clone-specific variations in membrane versus cytoplasmic staining patterns have been observed

• Flow Cytometry Applications:

  • Directly conjugated antibodies (e.g., PE or FITC-labeled) provide convenient single-step staining

  • Unconjugated antibodies with secondary detection systems often offer greater sensitivity

  • Clone 5D12 has demonstrated particular effectiveness for flow cytometry applications

• Emerging Technologies:

  • Synthetic antibody fragments (sFabs) like COP-1 show excellent specificity for human CLDN4 over other claudin family members

  • Bispecific antibodies like ASP1002 combine CLDN4 targeting with immune activation for enhanced therapeutic potential

What structural insights have been revealed about CLDN4 through recent research?

Recent structural studies have provided unprecedented insights into CLDN4:

• High-Resolution Structures: Using synthetic antibody fragments (sFabs), researchers have determined 2.2–2.6 Å structures of 22 kDa human CLDN-4 bound to 14 kDa cCpE using cryo-EM . These resolutions exceed all claudin/cCpE structures previously determined with X-ray crystallography.

• Binding Mechanisms: Structural analyses have revealed the precise interactions required for CpE-induced cytotoxicity and the unique mechanism of CLDN4-selective binding by synthetic antibody fragments like COP-1 .

• Platform Development: The development of a platform that synergizes structure determination and molecular targeting of claudins using sFabs has been a significant advancement in understanding CLDN4 structure and function .

• First Cryo-EM Structure: The work represents the first high-resolution structure of a claudin determined by cryo-EM, marking a technical breakthrough in claudin structural biology .

• Therapeutic Implications: These structural insights have uncovered potential new strategies to advance antibody-based therapies aimed at modulating tight junctions in tissue-specific ways through claudin-selective targeting .

How does CLDN4 expression vary across different cancer types and stages?

CLDN4 shows distinctive expression patterns across cancer types: