CDH3 Antibody

What is CDH3/P-cadherin and why is it a significant research target?

CDH3 (Cadherin-3), also known as P-cadherin, is a calcium-dependent cell-cell adhesion glycoprotein that plays crucial roles in cell adhesion, proliferation, and invasion processes. It has emerged as a significant research target because it's overexpressed in multiple cancer types including lung, breast, colorectal, pancreatic, head and neck, ovarian malignancies, and glioblastoma, while showing negligible expression in most normal tissues .

Research has demonstrated that CDH3 overexpression correlates with cancer aggressiveness, invasiveness, and poor patient prognosis . In glioblastoma, for example, CDH3 affects distinct cancer hallmarks in vitro and is related to increased tumor growth and shorter survival in vivo . Recent studies have identified CDH3 as a promising therapeutic target due to its selective expression pattern and involvement in oncogenic signaling pathways.

What applications are CDH3 antibodies typically used for in laboratory research?

CDH3 antibodies are employed across multiple laboratory techniques, with application specificity varying by clone and manufacturer. Common applications include:

For example, in IHC applications, CDH3 antibodies have been used to detect CDH3 in paraffin-embedded sections of human placenta tissues using heat-mediated antigen retrieval in citrate buffer (pH6) for 20 minutes . For ICC applications, CDH3 antibodies have been employed to detect the protein in A549 cells using enzyme antigen retrieval methods .

How should CDH3 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of CDH3 antibodies are critical for maintaining their specificity and sensitivity. Based on manufacturer recommendations:

• Store lyophilized antibodies at -20°C for up to one year from date of receipt

• After reconstitution, store at 4°C for one month or aliquot and store at -20°C for up to six months

• Avoid repeated freeze-thaw cycles which can significantly decrease antibody activity

• For long-term storage of reconstituted antibodies, glycerol addition (final concentration 15-50%) can help preserve activity

• Working dilutions should be prepared fresh before use for optimal results

• Follow specific manufacturer guidelines for each antibody clone, as storage conditions may vary slightly between products

Improper handling leading to denaturation or degradation is a common cause of experiment failure when working with antibodies.

How should researchers validate the specificity of CDH3 antibodies for their experimental model?

Validating CDH3 antibody specificity is crucial for ensuring experimental reproducibility and accuracy. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues with known CDH3 expression status. For example, studies have used A549 cells transfected with human or mouse CDH3 as positive controls and non-transfected A549 cells (which don't express CDH3) as negative controls .

• Genetic validation:

  • Use CRISPR/Cas9-engineered cell lines with CDH3 knockout

  • Compare siRNA-mediated CDH3 knockdown with control cells

  • Test in CDH3-overexpressing models versus parental cell lines

• Cross-reactivity testing: Validate specificity against related cadherins (CDH1/E-cadherin, CDH2/N-cadherin) to ensure the antibody doesn't cross-react with these structurally similar proteins .

• Multiple detection methods: Confirm expression using at least two independent methods (e.g., WB and IHC, or PCR and protein detection) .

• Epitope mapping: Understanding which region of CDH3 the antibody binds to can help predict potential cross-reactivity and application suitability. For example, some CDH3 antibodies target the extracellular domain while others target intracellular regions .

One published validation method used flow cytometry to examine the specificity of a CDH3 antibody, demonstrating a high correlation between mean fluorescence intensity values and expression levels of FLAG-tagged CDH3/P-cadherin in transformed cell lines .

What are the optimal immunohistochemistry protocols for detecting CDH3 in different tissue types?

Optimized IHC protocols for CDH3 detection vary by tissue type, fixation method, and antibody clone. Based on published methodologies:

For formalin-fixed paraffin-embedded (FFPE) tissues:

• Antigen retrieval options:

  • Heat-mediated retrieval in citrate buffer (pH 6.0) for 20 minutes (most common)

  • EDTA buffer (pH 8.0-9.0) for tissues with dense extracellular matrix

  • Enzyme-based retrieval for certain tissue types

• Blocking and antibody incubation:

  • Block with 10% goat serum (or serum matching secondary antibody species)

  • Primary antibody incubation: 1-5 μg/ml overnight at 4°C

  • Secondary detection: Biotinylated goat anti-rabbit/mouse IgG incubated for 30 minutes at 37°C

  • Signal development: Streptavidin-Biotin-Complex with DAB as chromogen

• Tissue-specific considerations:

  • Placental tissues show strong CDH3 membrane staining with minimal background

  • Tumor tissues may require titration optimization due to variable expression levels

  • Lung and colorectal cancer samples may benefit from dual staining with proliferation markers

For challenging tissues, a published protocol used enzyme antigen retrieval reagent for 15 minutes followed by blocking with 10% goat serum and overnight incubation with 1μg/ml anti-CDH3 antibody at 4°C, which yielded clear membrane staining in A549 cells .

What controls should be included when using CDH3 antibodies in research experiments?

Robust experimental design requires appropriate controls to validate findings and troubleshoot potential issues:

Essential controls for CDH3 antibody experiments:

• Positive tissue/cell controls:

  • Placental tissue (known high CDH3 expression)

  • CDH3-transfected cell lines (e.g., A549-CDH3)

  • Cancer cell lines with confirmed CDH3 expression (e.g., EBC1, H1373, SW948)

• Negative tissue/cell controls:

  • CDH3-negative cell lines (e.g., A549, RKO)

  • Normal tissues with minimal CDH3 expression

  • Isotype controls matched to primary antibody

• Technical controls:

  • Primary antibody omission control

  • Secondary antibody-only control

  • Blocking peptide competition assay using the immunizing peptide (if available)

  • Decreasing antibody concentration gradient to determine optimal signal-to-noise ratio

• Validation controls:

  • siRNA/shRNA knockdown versus scrambled control

  • CRISPR knockout versus wild-type cells

  • Parallel detection with a second CDH3 antibody recognizing a different epitope

For example, in published radioimmunotherapy studies, researchers confirmed specific binding of anti-CDH3 antibody by comparing tumor uptake in CDH3-positive tumors (EBC1, H1373, SW948) versus CDH3-negative tumors (A549, RKO), demonstrating significantly higher accumulation in CDH3-positive models .

How are CDH3 antibodies being developed for cancer therapeutic applications?

CDH3 antibodies have emerged as promising cancer therapeutic agents through several development strategies:

• Unconjugated antibodies with direct therapeutic effects:

  • PF-03732010, a fully human monoclonal antibody against P-cadherin, inhibits P-cadherin–mediated cell adhesion and aggregation in vitro

  • Demonstrated significant inhibition of tumor growth and metastatic progression in multiple CDH3-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, HCT116, H1650, PC3M-CDH3, and DU145

  • Mechanistically suppresses P-cadherin levels, causes degradation of membrane β-catenin, and concurrently suppresses cytoplasmic vimentin, resulting in diminished metastatic capacity

• Antibody-drug conjugates (ADCs):

  • BC3195, an ADC targeting CDH3 with monomethyl auristatin E (MMAE) payload, is in clinical development

  • Phase I clinical trial results show manageable safety profile with common adverse events including increased AST (55.6%), increased conjugated bilirubin (55.6%), and hypoalbuminemia (55.6%)

  • Preliminary antitumor activity observed with 50% of evaluable patients showing stable disease, including target lesion reduction in some cases

• Radioimmunotherapy approaches:

  • Yttrium-90 (90Y)-labeled anti-CDH3 mouse monoclonal antibody (MAb-6) demonstrated significant tumor suppression in CDH3-expressing cancer models

  • Single intravenous injection of 90Y-MAb-6 (100 μCi) significantly suppressed tumor growth in mice with CDH3-positive tumors

  • Two injections led to complete tumor regression in H1373-inoculated mice without detectable toxicity

• Bispecific antibody strategies:

  • TR2/CDH3 BAB, a human bispecific antibody that binds both CDH3 and TRAILR2, achieves TRAILR2 hyperclustering to induce apoptosis selectively in CDH3-expressing tumor cells

  • Demonstrated target-dependent anti-tumor activity in CRISPR/Cas9-engineered models

  • Employs an engineered human IgG1 Fc backbone with L234A/L235A mutations to avoid CDH3-independent crosslinking

These therapeutic approaches leverage CDH3's selective expression in cancer cells while minimizing off-target effects in normal tissues.

What are the most effective methods for quantifying CDH3 expression in patient samples for potential therapeutic targeting?

Accurate quantification of CDH3 expression is essential for patient selection in targeted therapy development. Multiple complementary approaches provide comprehensive assessment:

• Immunohistochemistry (IHC) with digital pathology:

  • Gold standard for clinical biomarker assessment

  • Membrane staining intensity typically scored on 0-3+ scale (0=negative, 1+=weak, 2+=moderate, 3+=strong)

  • H-score calculation: (% cells 1+ × 1) + (% cells 2+ × 2) + (% cells 3+ × 3), range 0-300

  • Digital pathology platforms enable objective quantification of membrane staining intensity and percentage positive cells

  • Requires standardized protocols with positive and negative controls to ensure reproducibility across laboratories

• Quantitative RT-PCR:

  • Enables precise quantification of CDH3 mRNA levels

  • Published protocols use TaqMan probes specific for CDH3 with TBP (TATA Box Binding Protein) as reference gene

  • PCR conditions: 2 min at 50°C, 20 s at 95°C, followed by 40 cycles of 3 s at 95°C and 30 s at 60°C

  • Results typically expressed as fold-change relative to control or as normalized expression units

• Proteomics approaches:

  • Mass spectrometry-based quantification of CDH3 protein expression

  • Provides absolute quantification independent of antibody affinity variations

  • Can simultaneously measure related proteins in cadherin pathways

  • More resource-intensive but offers higher specificity and broader pathway analysis

• Flow cytometry for circulating tumor cells or fresh tissue samples:

  • Enables single-cell analysis of CDH3 surface expression

  • Particularly useful for hematological samples or disaggregated solid tumors

  • Provides quantifiable data as mean fluorescence intensity (MFI)

  • Allows for multiparameter analysis with other cancer biomarkers

For clinical development of CDH3-targeted therapies, a combination of IHC and mRNA assessment has been most commonly employed, with positivity thresholds defined based on correlation with treatment response in early-phase clinical trials .

How do different CDH3 antibody clones compare in their binding properties and functional effects?

CDH3 antibody clones exhibit significant variations in binding properties and functional effects, which impact their research and therapeutic applications:

Functional differences between antibody clones arise from:

• Epitope specificity: Antibodies targeting different domains of CDH3 (extracellular, transmembrane, or cytoplasmic) exhibit varied functional effects. For example, antibodies targeting the extracellular domain may disrupt cell-cell adhesion, while others may primarily serve detection purposes .

• Affinity variations: Higher-affinity antibodies generally demonstrate superior sensitivity in detection applications but may not necessarily have superior functional effects in therapeutic applications.

• Isotype differences: The antibody isotype influences Fc-mediated functions such as complement activation and immune cell recruitment, which may contribute to therapeutic efficacy beyond target binding.

• Cross-reactivity profiles: Some antibodies cross-react with mouse CDH3, enabling preclinical studies in immunocompetent models, while others are strictly human-specific .

When selecting a CDH3 antibody clone, researchers should consider the specific application requirements and whether functional modulation or mere detection is the primary goal.

What are common technical challenges when working with CDH3 antibodies and how can they be overcome?

Researchers frequently encounter several technical challenges when working with CDH3 antibodies:

• Epitope masking in fixed tissues:

  • Challenge: Formalin fixation can mask CDH3 epitopes, reducing antibody accessibility

  • Solution: Optimize antigen retrieval methods; compare heat-induced (citrate pH 6.0 or EDTA pH 9.0) and enzymatic retrieval approaches. In published protocols, heat-mediated antigen retrieval in citrate buffer (pH6) for 20 minutes has proven effective for CDH3 detection in placental tissues .

• Membrane localization preservation:

  • Challenge: Preserving membrane staining pattern while minimizing cytoplasmic background

  • Solution: Use shorter fixation times (12-24h), optimize permeabilization conditions, and employ careful blocking procedures (10% serum matching secondary antibody species has shown good results) .

• Specificity concerns:

  • Challenge: Cross-reactivity with other cadherin family members (particularly CDH1/E-cadherin)

  • Solution: Use antibodies targeting unique CDH3 regions; perform validation in models with genetic manipulation of CDH3; include appropriate controls. The CDH3-building block of TR2/CDH3 BAB was derived from an immunization campaign using AlivaMab® transgenic mice and subsequently engineered to remove chemical liabilities and non-human residues .

• Signal amplification for low-expressing samples:

  • Challenge: Detecting CDH3 in samples with low expression levels

  • Solution: Employ signal amplification systems such as tyramide signal amplification or polymer-based detection systems; optimize primary antibody concentration and incubation time.

• Batch variability:

  • Challenge: Inconsistent results between antibody lots

  • Solution: Purchase larger quantities of validated lots; perform lot-to-lot validation; consider monoclonal antibodies for greater consistency.

• Species cross-reactivity limitations:

  • Challenge: Many CDH3 antibodies have limited cross-reactivity across species

  • Solution: Verify species reactivity before purchase; for cross-species studies, select antibodies targeting conserved epitopes or use species-specific antibodies. The MAb-6 antibody has demonstrated cross-reactivity with mouse CDH3/P-cadherin, making it suitable for preclinical studies in mouse models .

• Background in specific tissues:

  • Challenge: Non-specific binding in certain tissues (particularly liver)

  • Solution: Include additional blocking steps with avidin/biotin blocking for biotin-based detection systems; use Fc receptor blocking reagents; optimize antibody dilution through titration experiments.

Implementing these solutions based on specific experimental contexts will significantly improve CDH3 antibody performance across applications.

How can researchers optimize CDH3 antibody protocols for challenging tissue types or applications?

Optimizing CDH3 antibody protocols for challenging contexts requires systematic approach modifications:

• For highly fibrotic tissues (pancreatic cancer, breast cancer):

  • Extend antigen retrieval time to 30-40 minutes

  • Consider dual antigen retrieval: protease treatment followed by heat-induced retrieval

  • Section tissues at 3-4μm (thinner than standard) to improve antibody penetration

  • Use automated staining platforms for consistent results across samples

  • Employ polymer-based detection systems for higher sensitivity with lower background

• For circulating tumor cell detection:

  • Minimize processing time to preserve cell surface antigens

  • Use gentle fixation (2% paraformaldehyde for 10-15 minutes)

  • Block Fc receptors on leukocytes to reduce non-specific binding

  • Combine with epithelial markers (EpCAM) and exclude leukocyte markers (CD45)

  • Consider fluorescence-based detection for multiplexed analysis

• For multiplex immunofluorescence:

  • Test for antibody cross-reactivity with other targets in multiplex panel

  • Optimize stripping/quenching protocols between rounds if using sequential staining

  • Consider tyramide signal amplification for weaker signals

  • Use spectral unmixing to address autofluorescence in certain tissues

  • Include single-stain controls for each antibody to establish proper compensation

• For quantitative Western blot:

  • Optimize protein extraction for membrane proteins (consider NP-40 or Triton X-100-based lysis buffers)

  • Avoid boiling samples to prevent aggregation of transmembrane proteins

  • Use graduated loading controls to verify linear detection range

  • Consider transfer conditions optimized for high-molecular-weight proteins (91.4 kDa)

  • Use fluorescence-based detection systems for more accurate quantification

• For brain tissue analysis:

  • Optimize fixation time (24-48 hours maximum)

  • Perform antigen retrieval in Tris-EDTA buffer pH 9.0

  • Include Sudan Black treatment to reduce lipofuscin autofluorescence

  • Use confocal microscopy to better distinguish membrane staining patterns

  • Consider fresh frozen tissue for certain applications

One published optimization approach for ICC used enzyme antigen retrieval for 15 minutes with CDH3 antibody, followed by detection using Strepavidin-Biotin-Complex with DAB as the chromogen, which provided excellent visualization of CDH3 in A549 cells .

How should researchers interpret discrepancies between CDH3 mRNA and protein expression data?

Discrepancies between CDH3 mRNA and protein expression are common and have important implications for research interpretation:

• Common causes of discrepancies:

  • Post-transcriptional regulation: miRNAs can target CDH3 mRNA for degradation or translational inhibition

  • Protein stability differences: CDH3 protein half-life may vary between cell types or conditions

  • Technical limitations: Differences in sensitivity between mRNA detection methods (qPCR, RNA-seq) and protein detection methods (IHC, Western blot)

  • Antibody epitope accessibility: Protein conformation or interactions may mask antibody epitopes in certain contexts

  • Sample heterogeneity: Bulk analysis may mask cell-type specific expression patterns

• Systematic approach to resolving discrepancies:

  • Validation with multiple methods: Confirm protein expression using different antibody clones targeting distinct epitopes

  • Single-cell analysis: Employ single-cell RNA-seq paired with flow cytometry or single-cell proteomics

  • Temporal analysis: Assess whether differences reflect time-dependent regulation

  • Functional validation: Use genetic manipulation (knockdown/overexpression) to confirm specificity

  • Subcellular localization: Determine if protein is sequestered in specific cellular compartments

• Interpretation guidelines:

  • Protein expression is generally more relevant for functional studies and therapeutic targeting

  • mRNA expression may predict future protein expression or reflect recent transcriptional activity

  • In patient stratification, combined mRNA and protein assessment provides more robust classification

  • For antibody-based therapeutics, focus on protein expression and membrane localization

• Case study examples:

  • In glioblastoma research, CDH3 mRNA levels from TCGA database showed increased expression in high-grade gliomas, but protein confirmation was necessary to validate CDH3 as a therapeutic target

  • In pancreatic cancer, plasma membrane expression of CDH3 increases during carcinogenesis from benign pancreatic intraepithelial neoplasia 1 (PanIN-1) to malignant PDAC, highlighting the importance of assessing protein localization rather than just total levels

Researchers should ideally employ complementary approaches and consider the biological context when interpreting discrepancies between CDH3 mRNA and protein data.

How are CDH3 antibodies being utilized in combination therapeutic strategies for cancer treatment?

CDH3 antibodies are being integrated into innovative combination therapeutic strategies that exploit synergistic mechanisms:

• Combinations with conventional chemotherapies:

  • Preclinical studies have investigated CDH3-targeted therapies with standard-of-care cytotoxic drugs

  • TR2/CDH3 bispecific antibody has been evaluated in combination with indication-relevant cytotoxic drugs for pancreatic cancer

  • These combinations may enhance tumor penetration of chemotherapy through modulation of cell-cell adhesion

• Integration with immune checkpoint inhibitors:

  • CDH3-targeting may increase tumor immunogenicity by disrupting cell-cell contacts

  • Combination strategies with anti-PD-1/PD-L1 antibodies are being explored

  • Preliminary evidence suggests CDH3 inhibition may reduce immune-suppressive signals in the tumor microenvironment

• Antibody-drug conjugates with rationally selected payloads:

  • BC3195, an ADC targeting CDH3 with MMAE payload, represents a leading clinical-stage approach

  • Current phase I trial is exploring dosing up to 3.6 mg/kg with a BOIN design guiding dose escalation

  • Preliminary data shows manageable safety profile with 50% of evaluable patients showing stable disease

• Combination radiotherapy approaches:

  • 90Y-labeled anti-CDH3 antibody (90Y-MAb-6) demonstrated significant tumor suppression in CDH3-expressing cancer models

  • Two injections led to complete tumor regression in H1373-inoculated mice without detectable toxicity

  • Potential synergy with external beam radiation is being explored

• Bispecific and multispecific platforms:

  • TR2/CDH3 BAB bispecific antibody exploits dual targeting of CDH3 and TRAILR2 to achieve selective apoptosis induction in cancer cells

  • The engineered antibody uses silenced Fc domains (L234A/L235A mutations) to prevent CDH3-independent crosslinking

  • This approach extends the therapeutic concept previously applied with CDH17 to additional cancer indications with high medical need

These combination approaches aim to overcome resistance mechanisms, enhance efficacy, and broaden the therapeutic window for CDH3-targeted interventions.

What role do CDH3 antibodies play in understanding the biological functions of P-cadherin in normal and disease states?

CDH3 antibodies serve as crucial tools for elucidating P-cadherin biology across developmental, physiological, and pathological contexts:

• Developmental biology insights:

  • CDH3 antibodies have helped map expression patterns during embryonic development

  • Used to identify CDH3's role in placental morphogenesis and skin development

  • Enabled tracking of cell-cell adhesion dynamics during tissue formation

• Lineage tracing and cell fate determination:

  • P-cadherin expression marks specific epithelial progenitor populations

  • CDH3 antibodies facilitate isolation and characterization of these populations

  • Studies using CDH3 antibodies have revealed distinct stem/progenitor cell niches in various tissues

• Mechanistic studies in cancer progression:

  • CDH3 knockdown and overexpression GBM cell models revealed P-cadherin's oncogenic functions in affecting cell viability, cell cycle, invasion, migration, and neurosphere formation capacity

  • Genes positively correlated with CDH3 were found enriched for oncogenic pathways commonly activated in GBM

  • CDH3 antibodies helped demonstrate that GBM cells expressing high P-cadherin levels generate larger subcutaneous tumors and cause shorter survival in orthotopic models

• Epithelial-mesenchymal transition (EMT) research:

  • CDH3 antibodies used to monitor cadherin switching during EMT

  • PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, revealing potential mechanisms of its anti-metastatic effects

  • Immunofluorescence with CDH3 antibodies helped characterize these molecular changes during the transition

• Structure-function relationship exploration:

  • Domain-specific CDH3 antibodies help dissect the roles of different protein regions

  • Antibodies targeting the extracellular domain versus cytoplasmic domain reveal distinct functional effects

  • Epitope mapping with various antibody clones contributes to understanding critical functional domains

• Signaling pathway interrogation:

  • CDH3 antibody-based proximity ligation assays identify interaction partners

  • Immunoprecipitation with CDH3 antibodies enables identification of associated signaling complexes

  • Phospho-specific CDH3 antibodies detect activation states and regulatory mechanisms

What are the current limitations of CDH3 antibody research and potential future directions?

Despite significant progress, CDH3 antibody research faces several limitations that present opportunities for future innovation:

• Current technical limitations:

  • Limited availability of antibodies distinguishing between different CDH3 isoforms or post-translational modifications

  • Challenges in developing antibodies that specifically disrupt CDH3-mediated interactions with select binding partners

  • Insufficient characterization of epitope specificity for many commercially available clones

  • Restricted tools for real-time monitoring of CDH3 dynamics in living systems

• Biological knowledge gaps:

  • Incomplete understanding of CDH3's role in different cancer subtypes and progression stages

  • Limited insight into resistance mechanisms to CDH3-targeted therapies

  • Poorly characterized feedback mechanisms regulating CDH3 expression following therapeutic targeting

  • Unclear implications of heterogeneous CDH3 expression within tumors

• Future technical innovations:

  • Development of conformation-specific antibodies detecting active versus inactive CDH3 states

  • Creation of bispecific platforms targeting CDH3 together with complementary tumor-associated antigens

  • Engineering of antibody fragments (Fabs, scFvs) for improved tissue penetration

  • Generation of humanized or fully human antibodies with reduced immunogenicity for therapeutic applications

  • Design of intrabodies for targeting intracellular pools of CDH3

• Emerging research directions:

  • Exploration of CDH3's role in cancer stem cell maintenance and therapy resistance

  • Development of CDH3 antibody-based liquid biopsy approaches for minimally invasive disease monitoring

  • Investigation of CDH3-targeted radiotheranostic applications combining imaging and therapy

  • Creation of CDH3 CAR-T cell therapies for solid tumors

  • Characterization of CDH3's role in immune evasion and potential for enhancing immunotherapy response

• Translational challenges to address:

  • Determination of optimal patient selection biomarkers beyond mere CDH3 expression

  • Development of companion diagnostics with standardized scoring systems

  • Establishment of resistance mechanisms and strategies to overcome them

  • Optimization of dosing schedules and combination regimens

  • Exploration of potential synergies with emerging therapeutic modalities

The field is progressing toward more sophisticated understanding of CDH3 biology and therapeutic exploitation, with several clinical-stage assets including BC3195 (ADC) currently in Phase I trials and established preclinical evidence supporting various therapeutic modalities including radioimmunotherapy and bispecific antibody approaches .