Cdh17 Antibody

What is CDH17 and why are antibodies against it important in research?

CDH17 (cadherin 17) is a 92.2 kilodalton transmembrane protein that mediates cell-cell adhesion and is also known as CDH16, HPT-1, HPT1, HPT-1 cadherin, and LI cadherin . Antibodies against CDH17 are important research tools because CDH17 is frequently expressed in adenocarcinomas, including gastric cancer and hepatocellular carcinoma (HCC), and is associated with poor prognostic outcomes . These antibodies enable detection, quantification, and targeting of CDH17 in various experimental and potential therapeutic contexts.

What applications are CDH17 antibodies commonly used for in laboratory research?

CDH17 antibodies are utilized across multiple laboratory applications including:

• Western blotting (WB) for protein expression analysis

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

• Immunohistochemistry (IHC) for tissue localization

• Immunofluorescence (IF) and immunocytochemistry (ICC) for cellular visualization

• Flow cytometry (FCM) for quantitative cell surface expression analysis

The selection of antibody format should be based on specific experimental requirements, with consideration for clone specificity, reactivity across species, and conjugation options depending on the detection system employed.

How can researchers validate the specificity of CDH17 antibodies?

Validation of CDH17 antibody specificity can be performed through:

• Surface plasmon resonance analysis to confirm specific recognition of human CDH17, as demonstrated with the D2101 antibody that showed no cross-reactivity with murine CDH17

• Cell-based enzyme-linked immunosorbent assays to assess binding affinity and specificity

• Western blotting to confirm recognition of the correctly sized protein

• Use of CDH17 knockout or siRNA-treated cells as negative controls

• Comparative analysis with multiple antibody clones targeting different epitopes

• Immunoprecipitation followed by mass spectrometry to confirm target identity

What factors affect CDH17 antibody performance in different experimental systems?

Several factors can influence antibody performance when working with CDH17:

• Epitope accessibility: CDH17's transmembrane nature means antibodies targeting extracellular domains typically perform better in flow cytometry and live-cell applications, while those targeting intracellular domains require cell permeabilization

• Fixation methods: Some epitopes may be sensitive to certain fixatives; paraformaldehyde (4%) has been successfully used for CDH17 detection

• Species cross-reactivity: Many anti-CDH17 antibodies are human-specific with limited cross-reactivity to other species, necessitating careful selection for animal model studies

• Sample preparation: For membrane proteins like CDH17, proper sample preparation is critical to maintain protein conformation and epitope integrity

• Antibody concentration: Titration experiments are recommended as optimal concentrations vary by application

How should researchers design experiments to study CDH17 internalization dynamics?

To effectively study CDH17 internalization:

• Select antibodies that target extracellular domains of CDH17

• Culture cells on appropriate matrices (such as Matrigel-coated slides) that maintain proper cell morphology and CDH17 expression

• Implement pulse-chase protocols with fluorescently labeled antibodies to track internalization kinetics

• Use confocal microscopy with z-stack acquisition to visualize the internalization process

• Include appropriate controls such as temperature inhibition (4°C) to block active internalization

• Consider co-localization studies with endosomal and lysosomal markers to track intracellular trafficking

• For quantitative assessment, flow cytometry can measure surface vs. internalized antibody ratios over time

What are the optimal conditions for using CDH17 antibodies in flow cytometry?

For optimal flow cytometry results with CDH17 antibodies:

• Use non-enzymatic cell dissociation methods to preserve surface CDH17 integrity

• For detection of conformational changes in β1 integrin related to CDH17 function, use antibodies specific for the high-affinity conformation of β1 integrin with appropriate secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-mouse IgG)

• Perform careful titration of primary and secondary antibodies to determine optimal signal-to-noise ratios

• Include appropriate isotype controls and single-color controls for compensation

• When investigating CDH17-related signaling events, consider fixation and permeabilization protocols that preserve both surface CDH17 and intracellular phospho-proteins

• For multiparametric analysis, select fluorophores with minimal spectral overlap

How does CDH17 expression correlate with cancer progression and metastasis?

CDH17 has emerged as a significant biomarker in cancer progression:

• In hepatocellular carcinoma (HCC), CDH17 expression is associated with poor prognostic outcomes

• CDH17 has been identified as a marker in gastric cancer with potential utility for diagnosing primary lesions and lymph node metastasis

• Immunohistochemical analysis has revealed that CDH17 has a higher frequency of positivity in both primary and metastatic gastric cancer specimens compared to other markers such as HER2

• CDH17 is also highly expressed in colorectal cancer, often co-expressed with P-cadherin (pCAD), presenting opportunities for dual-targeting therapeutic approaches

This expression pattern makes CDH17 a valuable research target for understanding cancer progression mechanisms and developing diagnostic and therapeutic approaches.

What signaling pathways are modulated by anti-CDH17 antibodies in cancer cells?

Anti-CDH17 antibodies have been shown to modulate several critical signaling pathways:

• β-catenin signaling: The Lic5 monoclonal antibody markedly reduces CDH17 expression in a dose-dependent manner and suppresses β-catenin signaling in HCC cells

• Wnt pathway: Immunohistochemical and western blot analyses of xenograft explants treated with anti-CDH17 antibodies revealed inactivation of the Wnt pathway and suppression of Wnt signaling components in HCC tissues

• Apoptotic pathways: Anti-CDH17 antibodies induce cleavages of apoptotic enzymes caspase-8 and caspase-9 in HCC cells

• Integrin pathway: Some anti-CDH17 antibodies target RGD motifs that activate the α2β1integrin pathway, critical for cancer cell adhesion and invasion

Understanding these mechanisms is essential for developing targeted therapies and combination treatment strategies.

How can researchers evaluate the therapeutic efficacy of anti-CDH17 antibodies in preclinical models?

Robust evaluation of anti-CDH17 antibody therapeutic efficacy includes:

• In vitro assays:

  • Dose-dependent inhibition of CDH17 expression

  • Effects on cell proliferation, invasion, and migration

  • Analysis of downstream signaling pathway modulation

  • Apoptosis induction measurements

• In vivo models:

  • Subcutaneous xenograft models measuring tumor growth inhibition (TGI), as demonstrated with Lic5 antibody (5 mg/kg, i.p., t.i.w.) showing 60-65% TGI versus vehicle at day 28

  • Metastasis models, particularly lung metastasis which has shown marked suppression with anti-CDH17 antibody treatments

  • Combination therapy assessment, such as anti-CDH17 antibodies with conventional chemotherapy (e.g., cisplatin 1 mg/kg) showing enhanced efficacy (85-90% TGI)

  • Biodistribution studies using radiolabeled antibodies to assess tumor targeting specificity

What strategies exist for generating bispecific antibodies targeting CDH17 and other cancer markers?

Researchers have developed innovative approaches for bispecific antibody development:

• Avidity-driven in vitro screening approaches have successfully generated pCAD x CDH17 bispecific antibodies that selectively target cells expressing both antigens over cells expressing only one antigen

• Selection criteria should include in vitro binding assays and inhibition of cell proliferation results to identify lead bispecific candidates

• For antibody-drug conjugates (ADCs), bispecific antibodies can be linked to cytotoxic payloads such as monomethyl auristatin E (MMAE) to generate targeted therapeutic agents

• In vivo validation using dual flank mouse models allows demonstration of selective antitumor activity in tumors expressing both target antigens while sparing single-positive tissues

This dual-targeting approach represents a significant advancement in increasing the specificity of cancer therapeutics.

How can CDH17 antibodies be effectively radiolabeled for imaging and therapeutic applications?

Radiolabeling of anti-CDH17 antibodies requires careful consideration:

• Radionuclide selection: Indium-111 (111In) has been successfully used for imaging applications with anti-CDH17 antibodies such as D2101

• Conjugation chemistry: Methods should minimize impact on antibody affinity, as radiolabeling procedures have been observed to slightly decrease the affinity of some anti-CDH17 antibodies

• Quality control assessment: Surface plasmon resonance analysis can be used to evaluate whether radiolabeled antibodies maintain their specificity and binding properties

• Biodistribution studies: These are essential to confirm high uptake in target tissues (tumors) with low uptake in normal organs, including the stomach

• Imaging validation: SPECT/CT imaging with 111In-labeled anti-CDH17 antibodies should demonstrate high tumor-to-nontumor contrast ratios

What approaches can address the challenge of CDH17 species cross-reactivity limitations in preclinical models?

Addressing species cross-reactivity limitations requires strategic approaches:

• Epitope mapping and engineering: Identify conserved epitopes between human and model organism CDH17 for antibody development

• Humanized mouse models: Generate models expressing human CDH17 to overcome recognition issues, as some antibodies like D2101 specifically recognize human CDH17 but not murine CDH17

• Surrogate antibodies: Develop companion antibodies that target the corresponding epitope in the animal model's CDH17 for parallel preclinical studies

• Domain-specific targeting: Focus on highly conserved domains when cross-species reactivity is required

• Alternative model systems: Consider organoid cultures derived from human tissues that naturally express human CDH17

• Computational prediction: Use structural biology and in silico modeling to predict cross-reactive epitopes before antibody generation

How do anti-CDH17 antibodies targeting RGD motifs differ in mechanism from traditional antibodies?

Anti-CDH17 antibodies targeting RGD motifs represent a specialized approach:

• Mechanistic distinction: These antibodies specifically target the RGD motifs in cadherins that activate the α2β1integrin pathway, rather than simply binding to and blocking CDH17 function

• Functional impact: They can inhibit β1 integrin activation and subsequent signaling cascades that promote cancer cell adhesion, proliferation, and invasion

• Experimental validation: Flow cytometry assays using antibodies specific for β1 integrin in high-affinity conformation can confirm the mechanism of action

• Cross-cadherin effects: Similar approaches have been explored with other RGD-containing cadherins, including VE-cadherin domains 2 and 3 and CDH6

This mechanism-based targeting represents an advanced approach to therapeutic antibody development focusing on functional domains rather than simple protein recognition.

What are the challenges and solutions in developing CDH17 antibodies with dual diagnostic and therapeutic functions (theranostics)?

Developing effective CDH17 theranostics requires addressing several challenges:

• Challenges:

  • Maintaining antibody affinity and specificity after modifications for imaging or therapy

  • Achieving sufficient tumor accumulation for both imaging sensitivity and therapeutic efficacy

  • Determining optimal radioisotope selection for both diagnosis and therapy

  • Minimizing off-target effects in non-tumor tissues

• Solutions:

  • Site-specific conjugation technologies to preserve binding properties

  • Optimization of antibody fragments or alternative scaffolds for improved pharmacokinetics

  • Use of complementary radioisotope pairs (e.g., 111In for imaging and 177Lu for therapy) with similar chemistry

  • Pretargeting strategies to improve tumor-to-background ratios

  • Combination with predictive biomarkers to identify patients most likely to benefit

How can researchers integrate CDH17 antibody research with emerging technologies like single-cell analysis?

Integration with cutting-edge technologies offers new research avenues:

• Single-cell RNA sequencing can characterize CDH17 expression heterogeneity within tumors and correlate with response to anti-CDH17 therapies

• Mass cytometry (CyTOF) with anti-CDH17 antibodies enables simultaneous assessment of dozens of cellular parameters to understand CDH17's role in complex signaling networks

• Spatial transcriptomics combined with CDH17 immunohistochemistry can reveal microenvironmental influences on CDH17 expression and function

• CRISPR screening approaches can identify synthetic lethal interactions with CDH17, revealing new combination therapy opportunities

• Protein interaction mapping using proximity labeling with CDH17 antibodies can uncover novel binding partners and signaling nodes

• AI-based image analysis of CDH17 staining patterns may reveal subtle prognostic features not apparent through conventional analysis

What are common technical challenges when using CDH17 antibodies and how can they be addressed?

Researchers commonly encounter these challenges:

• Non-specific binding: Optimize blocking conditions with appropriate carriers (BSA, serum); validate with multiple negative controls

• Low signal intensity: Ensure antibody concentration is optimized; consider signal amplification methods; verify CDH17 expression levels in the sample

• High background in immunohistochemistry: Optimize antigen retrieval protocols; titrate primary and secondary antibodies; increase washing stringency

• Inconsistent results across experiments: Standardize protocols; use consistent antibody lots; implement positive control samples

• Loss of antigenicity during processing: Optimize fixation conditions; consider alternative fixatives; use fresh samples when possible

• Cross-reactivity issues: Validate antibody specificity with knockout/knockdown controls; consider more specific monoclonal alternatives

How can researchers optimize antibody-dependent cellular cytotoxicity (ADCC) with anti-CDH17 antibodies?

Optimizing ADCC activity requires systematic approach:

• Antibody engineering: Select appropriate isotypes (typically IgG1) or engineer Fc regions for enhanced FcγR binding

• Glycoengineering: Modify the glycosylation pattern of the Fc region (e.g., afucosylation) to enhance ADCC activity

• Effector cell considerations: Use optimal effector:target ratios in in vitro assays; consider the source and activation state of effector cells

• Combination strategies: Evaluate co-administration with cytokines that activate NK cells or other effector cells

• Assay development: Implement multiple complementary assay formats (chromium release, flow cytometry-based, bioluminescence) to comprehensively characterize ADCC activity

• In vivo assessment: Utilize immunocompetent models or humanized mouse models with reconstituted human immune system components

What considerations are important when developing competitive binding assays to characterize CDH17 antibody epitopes?

Effective competitive binding assays require:

• Reference antibody selection: Choose well-characterized antibodies with known epitopes as benchmarks

• Fragment preparation: Generate CDH17 fragments and peptides that isolate specific domains for epitope mapping

• Assay format selection: Surface plasmon resonance, ELISA, or flow cytometry-based competition assays each offer different advantages

• Controls: Include non-competing antibodies targeting distinct epitopes as negative controls

• Concentration optimization: Use appropriate concentration ranges to detect both high and low-affinity competition

• Data analysis: Apply appropriate mathematical models to determine if competition is complete or partial

• Confirmatory approaches: Validate findings with orthogonal methods such as hydrogen-deuterium exchange mass spectrometry or X-ray crystallography of antibody-antigen complexes