CLDN4 Recombinant Monoclonal Antibody

What is CLDN4 and why is it a significant target for monoclonal antibodies?

CLDN4 (Claudin-4) is a tetraspanin transmembrane protein crucial for tight junction formation and function in epithelial tissues. It plays essential roles in maintaining epithelial cell polarity and establishing intercellular barriers. CLDN4 serves as a well-known differentiation marker, with its presence indicating a more epithelial phenotype. Decreased expression of CLDN4 correlates with epithelial-mesenchymal transition (EMT), while high expression has been reported in multiple human malignancies, including ovarian, renal, and bladder cancers . These characteristics make CLDN4 an attractive target for monoclonal antibody development, particularly for cancer therapy applications where it's often overexpressed .

How are CLDN4 recombinant monoclonal antibodies typically produced?

Recombinant CLDN4 antibodies are produced through a multi-step process that begins with obtaining antibody genes. These genes are then cloned into a plasma vector to construct a vector clone, which is subsequently transfected into a mammalian cell line (typically CHO cells) for transient expression. The antibody is then purified using affinity chromatography techniques . In research settings, alternative approaches include immunizing mice (such as BXSB strain) with CLDN4-expressing cancer cell lines (e.g., Capan-2 pancreatic cancer cells), followed by hybridoma generation through fusion of murine splenocytes and myeloma cells. The hybridomas are then screened for specific binding to CLDN4 using cell-based screening methods .

What applications are CLDN4 recombinant monoclonal antibodies validated for?

CLDN4 recombinant monoclonal antibodies have been validated for multiple research applications including:

• Enzyme-linked immunosorbent assay (ELISA)

• Immunoprecipitation (IP)

• Western blotting (WB)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Flow cytometry

The specific applications depend on the epitope recognition properties of the antibody. For example, conformational epitope-recognizing antibodies may work well in applications where the protein maintains its native structure (IP, ELISA, flow cytometry) but may perform poorly in Western blotting where proteins are denatured .

How do I confirm the specificity of a CLDN4 monoclonal antibody?

To confirm specificity, multiple validation approaches should be employed:

• Cross-reactivity testing: Evaluate binding against related claudin family members (CLDN3, CLDN5, CLDN6, CLDN9) using overexpression systems such as transfected CHO cells .

• Knockout validation: Test the antibody in CLDN4 knockout samples to confirm absence of signal .

• Immunoprecipitation analysis: Verify that the antibody specifically precipitates CLDN4 protein of the expected molecular weight (approximately 22 kDa for native protein, 25 kDa for tagged versions) .

• Domain mapping: For advanced confirmation, use chimeric constructs where domains are exchanged between different claudin family members to identify the specific recognition epitope (e.g., extracellular loop 1 vs. loop 2) .

What strategies can optimize the design of anti-CLDN4 bispecific antibodies?

When designing bispecific antibodies targeting CLDN4 and another target molecule, consider these optimization strategies:

• Linker design: For single-chain variable fragments (scFv), the design of the linker between VH and VL domains is critical. Conventional Gly-Ser linkers (G₄S)ₙ provide conformational flexibility and minimal immunogenicity . The length of these linkers significantly impacts the antibody's structure and function:

  • Longer linkers (15-20 amino acids) promote the formation of monomeric scFvs

  • Shorter linkers (5-10 amino acids) encourage diabody formation

  • Very short linkers (<5 amino acids) can lead to triabody or tetrabody configurations

• Linker composition: Beyond standard glycine-serine linkers, consider adding charged residues like glutamic acid and lysine to enhance solubility .

• Domain orientation: Test both VH-VL and VL-VH orientations, as this can impact binding affinity and specificity to CLDN4.

• Format selection: For CLDN4 targeting, evaluate various bispecific formats (diabodies, TandAbs, knob-into-hole, etc.) based on the desired therapeutic mechanism—whether you need high tumor penetration (smaller formats) or extended half-life (larger formats containing Fc regions).

How can I determine if my anti-CLDN4 antibody recognizes conformational versus linear epitopes?

This distinction is crucial for application selection and experimental design:

• Comparative analysis approach:

  • Perform immunoprecipitation (IP) with native cell lysates

  • Conduct Western blotting (WB) with the same samples

  • If the antibody works in IP but fails in WB (as observed with KM3900), it likely recognizes a conformational epitope

• Epitope mapping strategy:

  • Create chimeric constructs by exchanging extracellular domains between CLDN4 and other claudins (e.g., CLDN6)

  • Examples of chimeric constructs:

    • CLDN4/6/6 (CLDN4/EL1 + CLDN6/EL2 + CLDN6/C-terminus)

    • CLDN6/4/4 (CLDN6/EL1 + CLDN4/EL2 + CLDN4/C-terminus)

    • CLDN4/4/6 (CLDN4/EL1&EL2 + CLDN6/C-terminus)

  • Express these in CHO cells and test antibody binding by flow cytometry

  • Binding patterns will reveal which domain contains the epitope

What factors affect the therapeutic efficacy of anti-CLDN4 antibodies in cancer treatment?

Several critical factors influence the therapeutic potential of anti-CLDN4 antibodies:

• Epitope location: Antibodies targeting the extracellular loop 2 (EL2) of CLDN4, like KM3900, show promise for therapeutic applications as this domain is accessible in intact cells .

• Effector functions: The antibody isotype determines its effector functions. Converting mouse IgG2a antibodies to chimeric human IgG1 (as with KM3934) can enhance:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Complement-dependent cytotoxicity (CDC)

• Tumor penetration: The size and format of the antibody affect tumor penetration. Some research suggests that for particular antibodies, dimeric or tetrameric forms may improve tumor targeting compared to monomeric forms .

• Cancer type-specific expression: CLDN4 expression varies across cancer types, with notable high expression in pancreatic and ovarian cancers. The level and pattern of expression will impact antibody efficacy .

• Claudin interaction network: CLDN4 interacts with other claudins in tight junctions, including CLDN3 and CLDN8. Understanding these interaction networks in specific cancer types may help predict antibody efficacy .

How do I design experiments to evaluate CLDN4 antibody-induced effects on tight junction dynamics?

To assess how anti-CLDN4 antibodies affect tight junction dynamics, consider these methodological approaches:

• Transepithelial electrical resistance (TEER) measurements:

  • Culture epithelial cells (e.g., MDCK, Caco-2) on permeable supports

  • Treat with anti-CLDN4 antibodies at various concentrations

  • Monitor changes in TEER over time to assess tight junction integrity

  • Compare results with isotype control antibodies

• Paracellular permeability assays:

  • Measure the flux of labeled molecules (fluorescent dextrans of varying sizes, lucifer yellow)

  • Determine if anti-CLDN4 antibodies affect size-selective permeability

  • This approach can reveal functional changes in tight junction strand dynamics

• Immunofluorescence microscopy:

  • Examine localization patterns of CLDN4 and other tight junction proteins (ZO-1, occludin)

  • Use live-cell imaging to track tight junction protein dynamics after antibody treatment

  • Quantify changes in junctional localization patterns

• Biochemical analysis of tight junction complexes:

  • Perform co-immunoprecipitation studies before and after antibody treatment

  • Assess changes in CLDN4 interactions with other claudins (CLDN3, CLDN8) or tight junction proteins

  • Analyze by Western blotting or mass spectrometry

This approach will provide insights into whether the antibody disrupts or modifies tight junction structure and function.

Why might my anti-CLDN4 antibody work in flow cytometry but not in Western blotting?

This discrepancy is commonly observed with antibodies that recognize conformational epitopes, as in the case of KM3900 . The explanation lies in protein structure preservation:

• Conformational epitope recognition: Some anti-CLDN4 antibodies recognize three-dimensional epitopes that are maintained in native protein configurations but lost during denaturation.

• Sample preparation differences:

  • Flow cytometry typically uses intact cells where membrane proteins maintain their native structure

  • Western blotting involves sample denaturation with SDS and heating, which disrupts protein folding

• Troubleshooting approaches:

  • For Western blotting: Try native (non-denaturing) PAGE instead of SDS-PAGE

  • Optimize fixation conditions: Some conformational epitopes may be preserved with milder fixatives

  • Consider partial denaturation or alternative detergents

  • Use alternative antibodies that recognize linear epitopes for Western blotting applications

What are the critical quality control parameters for CLDN4 recombinant monoclonal antibodies?

Ensure antibody quality through these essential parameters:

• Specificity assessment:

  • Cross-reactivity testing against related claudins (CLDN3, 5, 6, 9)

  • Validation in knockout/knockdown systems

  • Peptide competition assays if applicable

• Binding characteristics:

  • Determination of affinity constants (KD) using surface plasmon resonance

  • Assessment of on/off rates that might impact experimental outcomes

  • Epitope mapping to confirm recognition site

• Functionality testing:

  • Application-specific validation (IP, WB, IHC-P, flow cytometry)

  • Lot-to-lot consistency validation

  • Stability testing under various storage conditions

• Production quality:

  • Endotoxin levels (especially important for in vivo applications)

  • Aggregation assessment

  • Glycosylation pattern analysis for antibodies intended for functional studies

How can CLDN4 antibodies be utilized for studying tight junction dynamics in tissue barriers?

CLDN4 antibodies provide valuable tools for investigating tight junction biology:

• Visualization of tight junction remodeling:

  • Use non-blocking anti-CLDN4 antibodies conjugated to fluorescent proteins for live imaging

  • Track CLDN4 dynamics during junction assembly, disassembly, and remodeling

  • Combine with other labeled tight junction proteins to study co-localization patterns

• Barrier function modulation:

  • Identify antibodies that specifically disrupt CLDN4 interactions with other claudins

  • Use these to selectively perturb anion-selective channels formed by CLDN4-CLDN8 interactions

  • Measure resulting changes in paracellular ion selectivity

• Investigation of claudin strand interactions:

  • Apply antibodies targeting specific extracellular domains to block homophilic or heterophilic interactions

  • Study how CLDN4 integrates into CLDN3 strands to modulate tight junction properties

  • Examine how CLDN4 disrupts strand assembly of channel-forming CLDN2 and CLDN15

• Tissue-specific barrier regulation:

  • In renal collecting ducts, study CLDN4-CLDN8 co-assembly and its role in chloride permeability

  • In other epithelia, investigate tissue-specific claudin expression patterns and their functional significance

What considerations are important when using anti-CLDN4 antibodies for cancer immunotherapy research?

When investigating anti-CLDN4 antibodies as potential immunotherapeutics, consider:

• Therapeutic mechanism optimization:

  • Evaluate both direct and indirect mechanisms:

    • Direct: Blocking CLDN4 function in tumor cells

    • Indirect: Triggering immune effector mechanisms (ADCC, CDC)

  • Consider antibody engineering to enhance effector functions through Fc modifications

• Target accessibility assessment:

  • Evaluate CLDN4 expression and accessibility in tumor models

  • Consider that tight junction proteins may be less accessible in well-differentiated tumors

  • Determine if CLDN4 is redistributed to the cell surface in certain cancers, improving targeting

• Combination therapy approach:

  • Test anti-CLDN4 antibodies in combination with:

    • Traditional chemotherapeutics

    • Immune checkpoint inhibitors

    • Other targeted therapies

  • Assess potential synergistic effects

• Model selection considerations:

  • Use appropriate models that recapitulate CLDN4 expression patterns in human cancers

  • Consider patient-derived xenografts that maintain original tumor architecture

  • Evaluate efficacy in immunocompetent models if studying ADCC or CDC mechanisms