CEACAM5/CEACAM6 Recombinant Monoclonal Antibody

What are CEACAM5 and CEACAM6, and what is their biological significance?

CEACAM5 (also known as CEA) and CEACAM6 are members of the CEA-related cell adhesion molecules family, which comprises 18 genes and 11 pseudogenes. These are highly glycosylated proteins that function as intracellular and intercellular signaling molecules involved in cell differentiation, transformation, and immune response modulation .

CEACAM5 consists of one N-terminal variable domain and six C2-like immunoglobulin domains with a glycosylphosphatidylinositol (GPI) linker for membrane anchoring. It primarily functions as an adhesion molecule but also regulates differentiation, immune modulation, and inhibits anoikis (programmed cell death triggered by detachment from the extracellular matrix) .

CEACAM6 has an N-terminal variable domain followed by two C2-like immunoglobulin domains and is similarly anchored to the membrane via a GPI linker. It regulates anoikis through mechanisms involving Src and focal adhesion kinase (FAK) signaling pathways .

What is the expression pattern of CEACAM5/CEACAM6 in normal tissues versus cancer?

CEACAM5 and CEACAM6 show distinctive expression patterns across normal and cancerous tissues. In colorectal tumors matched with normal adjacent tissues, both CEACAM5 and CEACAM6 demonstrate significantly elevated expression in tumor tissues compared to normal tissues .

• Breast, pancreatic, colonic, and non-small-cell lung carcinomas

• Hyperplastic polyps and colon adenomas

• Mucinous ovarian cancer

• Gastric cancer

• Lung adenocarcinoma

CEACAM6 expression in pancreatic tumors varies by differentiation status, with the highest expression found in moderately differentiated tumors (7.5 ± 0.7), followed by moderately-poor differentiated (5.9 ± 1.9), well-moderately differentiated (5.8 ± 1.8), poorly-differentiated tumors (5.1 ± 2.5), and well-differentiated adenocarcinomas (4.0 ± 0.0). Non-neoplastic pancreas CEACAM6 expression is significantly lower at 2.25 ± 0.5 .

What methodologies are available for detecting CEACAM5/CEACAM6 in biological samples?

Several methodologies have been validated for detecting CEACAM5/CEACAM6 in biological samples:

• Enzyme-Linked Immunosorbent Assay (ELISA): Commercial ELISA kits are available with varying detection limits. For instance, induced sputum supernatant CEACAM6 can be measured using Sino Biological ELISA (detection range: 39.1-2500 pg/ml), while serum CEACAM6 is typically measured using R&D Systems ELISA (detection range: 0.625-20 ng/ml) .

• Immunohistochemistry (IHC): Tissue microarrays with specific antibodies targeting different domains can be used. For CEACAM6, antibodies like MN-15 and MN-3 targeting the A1B1- and N-domains respectively are employed, while for CEACAM5, antibodies like MN-14 targeting the A3B3 domain are utilized. The standard avidin-biotin-diaminobenzide staining protocol is commonly applied .

• Functional Binding Assays: The binding activity of antibodies against CEACAM5/CEACAM6 can be measured through functional ELISA, which evaluates the antibody's capacity to recognize and bind to the target antigen .

How do the structural characteristics of CEACAM5 and CEACAM6 influence antibody specificity and cross-reactivity?

The structural characteristics of CEACAM5 and CEACAM6 play crucial roles in determining antibody specificity and potential cross-reactivity. Recent cryo-electron microscopy studies have revealed important insights into the epitope-paratope interactions of antibodies targeting CEACAM5.

Tusamitamab, a selective antibody, specifically targets the A3-B3 domains of CEACAM5 without binding to related family members including CEACAM1, CEACAM6, or CEACAM8 . This selectivity is attributed to unique binding regions on the surface of these domains that confer antibody recognition and specificity.

The CEACAM family contains highly similar IgG-like domains, making the identification of unique epitopes critical for developing specific antibodies. Crystal structures are available for the N-terminal IgV-like domains of several CEACAM family members (CEACAM1 [PDB: 6XNO], CEACAM5 [PDB: 2QSQ], CEACAM6 [PDB: 4WHC], and CEACAM8 [PDB: 4YIQ]), but the structures of the A and B IgC2-like domains have only recently been elucidated .

Understanding the conformational constraints and unique epitopes is essential for rational design of highly specific antibodies that avoid off-target binding to structurally similar family members.

What is the functional relationship between CEACAM5 and CEACAM6 expression in cancer progression?

The functional relationship between CEACAM5 and CEACAM6 appears to be complex and interconnected. Research has demonstrated that when CEACAM6 expression is reduced through siRNA-mediated knockdown, CEACAM5 levels also decrease, suggesting an interdependence in their stability or expression regulation .

Both proteins have been implicated in cancer progression through various mechanisms:

• Inhibition of Differentiation: CEACAM5 has been shown to inhibit terminal differentiation. In the rat L6 myoblast cell line, stable CEACAM5 overexpression prevents fusion into myotubes and abrogates the molecular program of differentiation, including creatine phosphokinase upregulation, myogenin upregulation, and β-actin downregulation .

• Anoikis Resistance: Both proteins contribute to anoikis resistance, though through potentially different mechanisms. L6 rat myoblast cells transfected with CEACAM5 and CEACAM6 undergo significantly less anoikis than cells expressing CEACAM1 .

• Signaling Pathway Activation: While CEACAM6 activates the Src-FAK signaling system in a dose-dependent manner, leading to phosphorylation of MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase (ERK), CEACAM5 does not appear to affect this particular signaling pathway .

• Correlation with Disease Progression: In colorectal cancer, CEACAM6 levels are very low in stage I tumor samples but increase markedly in more advanced stages, suggesting a role in disease progression .

How do CEACAM5/CEACAM6 expression patterns vary across different cancer types and stages?

CEACAM5/CEACAM6 expression patterns exhibit significant variation across different cancer types and stages, with important implications for their use as biomarkers and therapeutic targets.

In lung cancers, adenocarcinomas express significantly more CEACAM6 than squamous carcinomas (P < 0.05), with varying expression patterns observed across different histological subtypes including well, moderately and poorly differentiated adenocarcinoma, squamous carcinoma, large cell, bronchioalveolar, large cell neuroendocrine, and small cell cancers .

In pancreatic cancer, CEACAM6 expression correlates with the degree of differentiation, with the highest expression observed in moderately differentiated tumors (7.5 ± 0.7), followed by moderately-poor (5.9 ± 1.9) and well-moderately differentiated (5.8 ± 1.8) tumors, with lower expression in poorly-differentiated tumors (5.1 ± 2.5) and well-differentiated adenocarcinomas (4.0 ± 0.0) .

In colorectal cancer, CEACAM6 levels show stage-dependent expression, with very low levels in stage I tumor samples but significant increases in more advanced stages. This pattern suggests a potential role in disease progression and metastasis .

The clinical relevance of these expression patterns is significant:

• CEACAM1 serves as a biomarker in melanoma, non-small cell lung cancer, and pancreatic adenocarcinoma, with increased expression associated with severe disease .

• CEACAM5 is an FDA-approved diagnostic tumor marker for colon cancer and has significant clinical utility as a tumor marker for gastrointestinal and respiratory malignancies, though its predictive value alone is limited by low sensitivity and specificity .

• CEACAM6 is highly expressed in multiple cancer types and may serve as a prognostic indicator in some contexts .

What are the optimal protocols for validating CEACAM5/CEACAM6 recombinant antibody specificity?

Validating the specificity of CEACAM5/CEACAM6 recombinant antibodies requires a multi-faceted approach to ensure accurate targeting and minimal cross-reactivity with other CEACAM family members. The following protocols represent best practices:

• ELISA Cross-Reactivity Testing: Test the antibody against recombinant proteins of all relevant CEACAM family members (particularly CEACAM1, CEACAM5, CEACAM6, and CEACAM8) to determine binding profiles and potential cross-reactivity. For example, the CEACAM5/CEACAM6 recombinant monoclonal antibody produced by Cusabio was validated using ELISA to confirm its reactivity with human CEACAM5 and CEACAM6 proteins .

• Epitope Mapping: Employ techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS), surface plasmon resonance (SPR), and cryo-electron microscopy to identify the specific epitopes recognized by the antibody. As demonstrated with tusamitamab, understanding the precise binding regions on the A3-B3 domains of CEACAM5 is critical for confirming specificity .

• siRNA Knockdown Experiments: Perform siRNA-mediated knockdown of CEACAM6 and assess the impact on CEACAM5 levels (and vice versa) to understand the relationship between these proteins and confirm antibody specificity in detecting changes in expression levels .

• Western Blotting with Cell Line Panels: Validate antibody specificity using Western blotting across a panel of cell lines with known varying expression levels of CEACAM5 and CEACAM6, including positive and negative controls.

• Immunohistochemistry Comparison: Compare staining patterns in patient-matched normal, primary tumor, and metastatic cancer specimens to confirm expected expression patterns and localization .

What considerations should be made when designing experiments using CEACAM5/CEACAM6 antibodies in different sample types?

When designing experiments with CEACAM5/CEACAM6 antibodies across different sample types, researchers should consider the following factors:

• Sample Preparation Effects:

  • For induced sputum samples, the effect of dithiothreitol (DTT) treatment should be assessed. Testing has shown that DTT does not affect the CEACAM6 standard curve in some ELISA assays .

  • Sample dilution may be necessary depending on expected antigen concentrations. Induced sputum samples often require dilution in assay diluent before ELISA .

• Detection Range Limitations:

  • Different commercial assays have varying detection limits. For instance, serum CEACAM6 measurement may require kits with higher detection limits (e.g., R&D Systems with 0.625-20 ng/ml) compared to sputum analysis (e.g., Sino Biological with 39.1-2500 pg/ml) .

• Antibody Format Selection:

  • Choose appropriately based on the experimental application. For CEACAM5/CEACAM6, unconjugated antibodies are available, as well as conjugated versions (Biotin, FITC, and HRP) .

• Storage and Stability:

  • Maintain antibodies at 2-8°C for frequent use or -20°C for extended storage (up to 12 months), avoiding repeated freeze/thaw cycles that may compromise activity .

• Tissue-Specific Expression Levels:

  • Be aware that expression levels vary significantly between normal and cancerous tissues, as well as between different cancer types and stages. This may necessitate adjustments in antibody concentration or detection methods .

• Applications Compatibility:

  • Verify that the selected antibody has been validated for the intended application. For example, some CEACAM5/CEACAM6 antibodies are specifically validated for ELISA but may require additional validation for other applications like IHC or Western blotting .

How can researchers accurately quantify CEACAM5/CEACAM6 expression in heterogeneous tumor samples?

Accurately quantifying CEACAM5/CEACAM6 expression in heterogeneous tumor samples presents unique challenges that require specialized approaches:

• Tissue Microarray (TMA) Analysis with Scoring Systems:

  • Implement standardized scoring systems (e.g., 0-8 scale) to evaluate expression intensity across different histological regions.

  • Record the average score ± standard deviation for each histotype to account for intratumoral heterogeneity .

• Multiple Domain-Specific Antibodies:

  • Use antibodies targeting different domains of the proteins for comprehensive detection. For example, MN-15 and MN-3 antibodies target the A1B1- and N-domains of CEACAM6 respectively, while MN-14 targets the A3B3 domain of CEACAM5 .

• Single-Cell RNA Sequencing:

  • Apply single-cell RNA-seq to delineate expression patterns at the cellular level within heterogeneous tumors, aligning with previously published data that has revealed variable expression across cell populations .

• Digital Pathology and Image Analysis:

  • Employ digital pathology tools with machine learning algorithms to quantify expression levels across entire tumor sections, accounting for spatial heterogeneity.

• Laser Capture Microdissection:

  • Use laser capture microdissection to isolate specific tumor regions for more precise expression analysis in areas of interest.

• Patient-Matched Analysis:

  • When possible, analyze patient-matched normal, primary tumor, and metastatic specimens to establish baseline expression and track changes throughout disease progression .

• Consideration of Tumor Differentiation Status:

  • Stratify analysis based on tumor differentiation status, as expression levels can vary significantly. For example, in pancreatic cancer, moderately differentiated tumors show higher CEACAM6 expression (7.5 ± 0.7) compared to well-differentiated tumors (4.0 ± 0.0) .

How should researchers interpret conflicting CEACAM5/CEACAM6 expression data across different detection methods?

When confronted with conflicting CEACAM5/CEACAM6 expression data across different detection methods, researchers should consider the following interpretative framework:

• Method-Specific Sensitivity and Dynamic Range:

  • Different detection methods have varying sensitivities. For example, ELISA may detect soluble forms of the proteins, while IHC detects membrane-bound forms. ELISA systems themselves have different detection limits; serum CEACAM6 measurement kits (0.625-20 ng/ml) have higher detection limits than those for sputum analysis (39.1-2500 pg/ml) .

• Epitope Accessibility:

  • Different antibodies target distinct domains of CEACAM5/CEACAM6. The accessibility of these epitopes may vary depending on protein conformation, glycosylation status, or protein-protein interactions, leading to apparent differences in expression .

• Sample Processing Effects:

  • Sample preparation methods can affect detection. Researchers should verify whether sample treatments (e.g., DTT in sputum samples) impact assay performance through spiking experiments with known concentrations of CEACAM6 across the assay range .

• Specific vs. Cross-Reactive Detection:

  • Some antibodies, like tusamitamab, specifically target CEACAM5 without binding to CEACAM6 , while others may detect both proteins or cross-react with other family members. Confirm the specificity profile of the antibodies used.

• Correlation Analysis Between Methods:

  • Perform correlation analysis between different detection methods on the same samples to establish method-specific correction factors.

• Biological Versus Technical Variation:

  • Heterogeneous expression within tumors is a biological reality, as evidenced by observations that some tumor areas show overlap between CEACAM5 and CEACAM6, while others do not . Distinguish this from technical variability.

What are the common pitfalls in experimental design when studying CEACAM5/CEACAM6 interactions?

Researchers should be aware of several common pitfalls when designing experiments to study CEACAM5/CEACAM6 interactions:

• Neglecting Functional Interdependence:

  • Failure to account for the functional relationship between CEACAM5 and CEACAM6. Evidence suggests that knockdown of CEACAM6 leads to decreased CEACAM5 levels, indicating interdependence in their expression or stability .

• Overlooking Isotype Controls:

  • Not including appropriate isotype controls (e.g., hIgG1 for humanized antibodies) can lead to misinterpretation of binding specificity .

• Incorrect Domain Targeting:

  • Targeting the wrong domains for specific applications. The A3-B3 domains of CEACAM5 contain unique binding regions that confer specificity, whereas targeting more conserved domains might result in cross-reactivity with other CEACAM family members .

• Disregarding Cancer Stage and Differentiation:

  • Failing to stratify analysis based on cancer stage and differentiation status. CEACAM6 levels are very low in stage I colorectal tumor samples but increase markedly in more advanced stages . Similarly, expression varies with differentiation status in pancreatic tumors .

• Inadequate Consideration of Glycosylation:

  • Not accounting for glycosylation patterns, which can affect antibody recognition. Both CEACAM5 and CEACAM6 are highly glycosylated proteins, and glycosylation patterns may vary across different cell types and disease states .

• Single Detection Method Reliance:

  • Relying on a single detection method without validation through complementary approaches. The epitopes and paratopes identified by different methods (HDX-MS, SPR, and cryo-EM) may show general agreement but also some differences .

How can researchers assess the functional impact of CEACAM5/CEACAM6 targeting in experimental models?

Assessing the functional impact of CEACAM5/CEACAM6 targeting requires comprehensive experimental approaches that evaluate multiple cellular processes:

• Anoikis Resistance Assays:

  • Measure anoikis (detachment-induced apoptosis) in cell lines with and without antibody treatment. Both CEACAM5 and CEACAM6 have been shown to inhibit anoikis, though potentially through different mechanisms .

• Signaling Pathway Analysis:

  • Evaluate the impact on key signaling pathways, particularly:

    • Src-FAK signaling system (activated by CEACAM6 in a dose-dependent manner)

    • MAPK/MEK1/2 and ERK phosphorylation status

    • Note that while CEACAM6 activates these pathways, CEACAM5 may not affect Src-FAK signaling .

• Differentiation Program Assessment:

  • Analyze markers of cell differentiation such as creatine phosphokinase, myogenin, and β-actin, as CEACAM5 has been shown to abrogate the molecular program of differentiation .

• Cell Adhesion and Migration Studies:

  • Investigate the effects on cell adhesion properties and migratory capacity, given the role of these proteins as adhesion molecules .

• In Vivo Tumor Growth and Metastasis Models:

  • Employ animal models to assess the impact of antibody treatment on tumor growth, invasion, and metastasis.

  • Consider patient-derived xenograft (PDX) models to maintain the heterogeneity and complexity of human tumors.

• Combination Treatment Approaches:

  • Evaluate the effects of combining CEACAM5/CEACAM6-targeting antibodies with standard chemotherapeutic agents or other targeted therapies to identify potential synergistic interactions.

• Cross-Family Member Analysis:

  • Assess the impact of CEACAM5/CEACAM6 targeting on the expression and function of other CEACAM family members, given their potential compensatory mechanisms.