CEACAM5 Recombinant Monoclonal Antibody

What is CEACAM5 and why is it a significant target for cancer research?

CEACAM5, also known as CD66e or CEA, is the founding member of the carcinoembryonic antigen (CEA) family of proteins within the immunoglobulin (Ig) superfamily. Its significance stems from several key characteristics:

• CEACAM5 is synthesized during fetal gut development and is re-expressed in increased amounts in various malignancies, particularly intestinal carcinomas

• The extracellular domains of CEACAM5 function as:

  • Homophilic and heterophilic intercellular adhesion molecules

  • Human pathogen receptors

• CEACAM5 expression has been documented in multiple solid tumors including:

  • Colorectal adenocarcinoma (primary expression site)

  • Lung adenocarcinoma (60-70% are CEACAM5+)

  • Gastric adenocarcinoma

  • Esophageal adenocarcinoma

  • Pancreatic carcinoma

  • Gallbladder adenocarcinoma

  • Urachal carcinoma

  • Salivary gland tumors

  • Ovarian and endocervical adenocarcinomas

This expression profile makes CEACAM5 a valuable diagnostic marker and therapeutic target, particularly for differentiating adenocarcinomas from other malignancies like pleural mesotheliomas (which rarely or weakly express CEACAM5) .

How do recombinant monoclonal anti-CEACAM5 antibodies differ from conventional antibodies in research applications?

Recombinant monoclonal anti-CEACAM5 antibodies offer several advantages over conventional (hybridoma-derived) antibodies:

• Improved consistency: Recombinant antibodies show higher repeatability and batch-to-batch consistency

• Increased sensitivity: Enhanced detection capabilities for low-abundance CEACAM5 expression

• Confirmed specificity: Better discrimination between CEACAM5 and other family members like CEACAM1, CEACAM6, and CEACAM8

• Production flexibility: Can be produced in various expression systems (e.g., HEK293 cells)

• Sequence-defined paratopes: Enable detailed epitope mapping studies for therapeutic development

For example, the CC4 anti-CEACAM5 monoclonal antibody demonstrates excellent specificity for CEACAM5 in flow cytometry, immunofluorescence, and immunohistochemistry applications, with minimal cross-reactivity to other family members .

What are the optimal protocols for using anti-CEACAM5 antibodies in immunohistochemistry?

For optimal immunohistochemical staining with anti-CEACAM5 antibodies, researchers should consider:

Sample preparation:

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

  • Some antibodies (e.g., COL-1 clone) require antigen retrieval by boiling tissue sections in 10mM Tris Buffer, pH 8.5-9.0 for 10-20 minutes followed by cooling at room temperature for 20 minutes

  • Typical section thickness: 4-6 μm

Protocol recommendations:

• Working dilution: 0.5-2 μg/mL for most commercial anti-CEACAM5 antibodies

• Incubation: 30 minutes at room temperature

• Detection system: Polymer-based detection systems are preferred for enhanced sensitivity

• Controls:

  • Positive control: Colorectal adenocarcinoma tissue

  • Negative control: Non-epithelial tissues (confirm antibody specificity)

Expected results:

• Positive staining patterns: Cytoplasmic and luminal membrane localization in adenocarcinoma cells

• Negative tissues: Most benign glands, stroma, and non-epithelial malignancies should show no staining

The staining pattern can help evaluate tumor differentiation and heterogeneity, with stronger membranous staining typically observed in well-differentiated adenocarcinomas.

How can researchers effectively employ anti-CEACAM5 antibodies in flow cytometry applications?

For effective flow cytometry applications with anti-CEACAM5 antibodies:

Protocol optimization:

• Cell preparation:

  • Single-cell suspension preparation is critical (enzymatic dissociation may affect some epitopes)

  • Fixation: If needed, use 1-4% paraformaldehyde for 10-15 minutes at room temperature

• Antibody concentration: 0.5-1 μg per 10^6 cells

• Buffer composition: PBS with 0.5-2% BSA and 0.05-0.1% sodium azide

• Incubation conditions: 30 minutes at 4°C

Gating strategy:

• Forward/side scatter to exclude debris and select intact cells

• Live/dead discrimination using appropriate viability dyes

• Isotype controls to set negative populations

Validation approach:

• Transfected vs. non-transfected cells as controls

  • As demonstrated with anti-CEACAM5 (DM120) on Expi293 cells transfected with human CEACAM5 versus Expi293 cells transfected with irrelevant protein

• Comparison with known CEACAM5-positive cell lines (e.g., LS174T, SW1116)

Data analysis considerations:

• Mean fluorescence intensity (MFI) for quantitative comparison

• Percent positive cells for heterogeneous populations

• Compensation for spectral overlap when using multiple fluorophores

This approach enables accurate detection and quantification of CEACAM5 expression levels across different cell populations.

What methodology should be used to validate the specificity of anti-CEACAM5 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For anti-CEACAM5 antibodies, a comprehensive validation approach includes:

1. Molecular characterization:

• Western blot analysis under reduced and non-reduced conditions

  • Example: CC4 mAb shows differential binding patterns under these conditions, confirming conformation-dependent epitope recognition

• Immunoprecipitation followed by mass spectrometry

  • As demonstrated with CC4 mAb, which identified four CEACAM5-specific peptides

2. Cell-based validation:

• Transfection studies:

  • Overexpression of CEACAM5 in non-expressing cell lines

  • Example: Bladder cancer T2-4 cells transfected with CEACAM5 expression plasmids showed positive staining with CC4 antibody

• Knockout/knockdown approaches to confirm signal loss

3. Cross-reactivity assessment:

• Testing against other CEACAM family members

  • Important for verifying that antibodies don't recognize CEACAM1, CEACAM6, or CEACAM8

• Epitope mapping studies:

  • Using recombinant CEACAM5 fragments and deletion mutants

  • Example: CC4 epitope mapped to aa42-61 region

4. Tissue validation:

• Immunohistochemistry on known positive and negative tissues

• Comparison of staining patterns with established CEACAM5 expression profiles

5. Functional validation:

• Confirming biological effects align with CEACAM5 mechanisms

  • Example: CC4 mAb suppresses colorectal tumor cell proliferation, migration, and aggregation in a dose-dependent manner

This multi-platform validation approach ensures confident interpretation of experimental results with anti-CEACAM5 antibodies.

What are the critical structural features of CEACAM5 that influence antibody binding and specificity?

The structural features of CEACAM5 that impact antibody binding include:

Domain architecture:

Key binding regions:

• The N-terminal domain (aa35-155) contains epitopes for many antibodies

  • CC4 antibody specifically binds to a narrow region (aa42-61) in this domain

• The A3-B3 domains (aa499-685) harbor epitopes for other antibodies

  • Tusamitamab specifically targets this region and doesn't bind to other CEACAM family members

Glycosylation patterns:

• CEACAM5 contains multiple N-linked glycosylation sites that influence antibody recognition

• High-resolution structural studies have included these glycans in epitope mapping

Conformational epitopes:

• Some antibodies recognize conformational epitopes dependent on proper protein folding

  • Evidenced by differential binding under reduced versus non-reduced conditions

Unique binding determinants:

• Specific amino acid sequences distinguish CEACAM5 from other family members

  • For example, the region aa42-61 in CEACAM5 differs from the corresponding region in CEACAM1

These structural characteristics enable the development of highly specific antibodies that can discriminate between CEACAM5 and closely related family members.

How do anti-CEACAM5 monoclonal antibodies like CC4 suppress tumor growth mechanistically?

Anti-CEACAM5 antibodies suppress tumor growth through multiple mechanisms, as demonstrated with the CC4 antibody:

1. Direct cellular effects:

• Inhibition of proliferation:

  • CC4 treatment reduces cell division in colorectal cancer cell lines (LS174T and Lovo)

  • Demonstrated using CFSE staining in dose-dependent manner

• Suppression of cell migration:

  • CC4 inhibits cell motility in chamber assays

  • Dose-dependent effect observed in multiple colorectal cancer cell lines (LS174T, Lovo, Colo-205)

• Disruption of cell aggregation:

  • CC4 prevents cancer cell aggregation in a calcium-dependent manner

  • At 50 μg/ml, approximately 80% of LS174T cells remained single, compared to only 40% with control IgG

2. Immune modulation effects:

• Enhancement of NK cell-mediated cytotoxicity:

  • CC4 disrupts the inhibitory CEACAM5-CEACAM1 interaction between tumor cells and NK cells

  • This prevents the immune evasion mechanism normally triggered by this interaction

  • Demonstrated in experiments with both CD56+CD16+ and CD56+CD16- NK cells

• Blockade of CEACAM1 inhibitory signaling:

  • CEACAM1 contains ITIM (immunoreceptor tyrosine-based inhibition motif) sequences

  • When engaged by CEACAM5, these motifs trigger inhibitory signaling in NK cells

  • CC4 prevents this engagement, restoring NK cell activity against tumor cells

3. In vivo tumor suppression:

• Xenograft studies show CC4 specifically accumulates at tumor sites

• Remarkable repression of colorectal tumor growth observed in these models

This multi-modal mechanism of action makes anti-CEACAM5 antibodies promising therapeutic candidates for colorectal and other CEACAM5-expressing cancers.

What methodologies are used for epitope mapping of anti-CEACAM5 antibodies?

Epitope mapping of anti-CEACAM5 antibodies employs multiple complementary approaches:

1. Recombinant domain/fragment analysis:

• Domain-level mapping:

  • Expression of individual CEACAM5 domains (N, A1, B1, A2, B2, A3, B3)

  • Example: CC4 antibody epitope was initially localized to Domain1 (aa35-155)

• Progressive truncation strategy:

  • Creation of fragments with systematic truncations

  • CC4 epitope was further narrowed to aa42-61 through testing fragments F42-273, F62-273, F80-273, and F101-273

2. Alanine scanning mutagenesis:

• Systematic amino acid substitution:

  • Key residues within epitopes are individually replaced with alanine

  • Example: For tusamitamab, five variants in the heavy chain (Asp33Ala, Phe101Ala, Gly102Ala, Gly105Ala, and Pro106Ala) and two in the light chain (Tyr32Ala and Asn50Ala) exhibited complete loss of binding to CEACAM5

3. Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Measures the rate of hydrogen-deuterium exchange in peptide backbones

• Regions protected from exchange upon antibody binding indicate epitope locations

• Provides detailed insights into conformational epitopes

4. Structural biology approaches:

• Cryo-electron microscopy (cryo-EM):

  • High-resolution structure determination of antibody-antigen complexes

  • Example: Structure of tusamitamab bound to the A3-B3 domains of CEACAM5

• X-ray crystallography:

  • Atomic-level resolution of antibody-antigen interfaces

  • Particularly useful for rigid epitopes

5. Competitive binding assays:

• Evaluates whether different antibodies compete for the same epitope region

• Useful for comparing novel antibodies to those with known epitopes

These methods collectively provide comprehensive mapping of epitope-paratope interactions, informing rational design of improved CEACAM5-targeting therapeutics.

How can researchers leverage anti-CEACAM5 antibodies to study NK cell-mediated tumor immunity?

Anti-CEACAM5 antibodies offer unique tools for investigating NK cell-tumor interactions:

1. Experimental models for studying CEACAM5-CEACAM1 interactions:

• Cell line system development:

  • Transfection of CEACAM5-negative cancer cells (e.g., HCT-15) with CEACAM5 or CEACAM1 expression vectors

  • Creation of stable cell lines (HCT-15/CEACAM5 and HCT-15/CEACAM1)

  • Verification of expression using flow cytometry

• NK cell preparation:

  • Isolation of CD56+CD16+ and CD56+CD16- NK cell subsets

  • Confirmation of CEACAM1 expression on NK cells

2. NK cytotoxicity assay methodologies:

• Tumor target cell preparation:

  • CEACAM5-expressing colorectal cancer cells (endogenous or transfected)

• Effector:target ratio optimization:

  • Testing multiple E:T ratios to determine optimal assay sensitivity

  • Example study used varying ratios to demonstrate CEACAM5-dependent effects

• Cytotoxicity measurement:

  • 51Cr-release assays or flow cytometry-based methods

• Antibody blockade experiments:

  • Addition of anti-CEACAM5 antibodies (e.g., CC4) at defined concentrations

  • Inclusion of isotype controls for comparison

3. Signaling pathway analysis:

• ITIM-dependent signaling investigation:

  • Analysis of tyrosine phosphorylation in CEACAM1's cytoplasmic tail

  • Evaluation of downstream inhibitory signaling cascade components

• Molecular intervention approaches:

  • CEACAM1 mutants lacking ITIM motifs

  • SHP-1/SHP-2 phosphatase inhibitors

4. In vivo models:

• Humanized mouse systems:

  • Mice engrafted with human NK cells and CEACAM5+ tumors

  • Treatment with anti-CEACAM5 antibodies

  • Monitoring tumor growth and NK cell infiltration

This research framework has revealed that CEACAM5-CEACAM1 interactions represent an immune evasion mechanism in colorectal cancer, with CEACAM5 on tumor cells engaging CEACAM1 on NK cells to suppress cytotoxicity. Anti-CEACAM5 antibodies like CC4 can disrupt this interaction, potentially restoring anti-tumor immunity .

What approaches can address cross-reactivity issues with other CEACAM family members?

Cross-reactivity with related CEACAM family members presents a significant challenge in developing specific anti-CEACAM5 antibodies. Researchers can employ several strategies to address this issue:

1. Epitope-focused antibody design:

• Unique epitope identification:

  • Comparative sequence analysis of CEACAM family members

  • Targeting regions with lowest homology (e.g., aa42-61 region for CC4)

• Structure-guided approach:

  • Utilizing high-resolution structures of CEACAM5-specific domains

  • Example: A3-B3 domains targeted by tusamitamab show unique structural features compared to other family members

2. Comprehensive screening methodologies:

• Multi-platform validation:

  • Testing candidate antibodies against cells expressing different CEACAM family members

  • Example: Flow cytometry analysis using transfected cells expressing individual CEACAM proteins

• Sequential absorption:

  • Pre-absorbing antibodies with recombinant non-target CEACAM proteins

  • Testing residual binding to CEACAM5

3. Mutation and chimeric protein approaches:

• Domain swapping experiments:

  • Creating chimeric proteins with domains exchanged between CEACAM family members

  • Identifying which domains confer specificity

• Site-directed mutagenesis:

  • Converting specific amino acids in CEACAM5 to corresponding residues in other family members

  • Example: Mutation studies comparing region aa42-61 of CEACAM1 and CEACAM5

4. Negative selection strategies:

• Counter-screening:

  • Eliminating antibody candidates showing binding to CEACAM1, CEACAM6, or CEACAM8

  • Prioritizing antibodies with >100-fold selectivity for CEACAM5

These approaches have successfully yielded highly specific antibodies like CC4 and tusamitamab that discriminate between CEACAM5 and other family members, enabling both precise research applications and targeted therapeutic development.

How can researchers optimize storage and handling of anti-CEACAM5 recombinant antibodies to maintain functionality?

Proper storage and handling are critical for maintaining antibody functionality. For anti-CEACAM5 recombinant antibodies:

1. Formulation considerations:

• Buffer composition:

  • Typically supplied in phosphate-buffered saline (PBS), pH 7.4

  • Some formulations include 5-8% trehalose as a cryoprotectant

• Protein stabilizers:

  • Addition of 0.1% human serum albumin (HSA) recommended for long-term storage after reconstitution

• Preservatives:

  • Sodium azide (0.05-0.1%) may be included for prevention of microbial growth

  • Note: Azide can interfere with some applications (e.g., cell culture)

2. Storage conditions:

• Temperature requirements:

  • Lyophilized form: Stable for 1 week at room temperature; store at -20°C to -80°C for 12 months

  • Reconstituted: Store at 4°C for 2-7 days or below -20°C for up to 3 months

• Freeze-thaw considerations:

  • Limit freeze-thaw cycles to 3-5 maximum

  • Aliquot antibodies before freezing to avoid repeated freeze-thaw cycles

3. Reconstitution protocols:

• Recommended procedure:

  • Reconstitute lyophilized antibody with sterile water to 100 μg/ml

  • Solubilize at room temperature for 30 minutes with occasional gentle mixing

• Critical precautions:

  • Avoid vigorous vortexing (can cause denaturation)

  • Centrifuge briefly after reconstitution to collect solution

4. Quality control approaches:

• Functionality testing:

  • Validate activity periodically using established applications (ELISA, flow cytometry)

  • Example: ELISA binding to recombinant CEACAM5 protein in the 0.1-50 ng/ml range

• Appearance monitoring:

  • Check for visible precipitation or cloudiness before use

  • Filter if necessary using low protein-binding filters

These practices help ensure consistent performance of anti-CEACAM5 recombinant antibodies across experimental applications, enabling reliable research outcomes.

What are the emerging applications of anti-CEACAM5 antibodies in targeted cancer therapeutics?

Anti-CEACAM5 antibodies are being explored in multiple innovative therapeutic approaches:

1. Antibody-drug conjugates (ADCs):

• Current development:

  • Tusamitamab ravtansine (SAR408701) combines anti-CEACAM5 antibody with a cytotoxic payload

  • Investigated for treatment of advanced nonsquamous non-small cell lung cancer and other solid tumors

• Mechanistic considerations:

  • The epitope location on A3-B3 domains is important for invagination of the cell membrane, which affects ADC internalization and efficacy

2. Bispecific antibodies:

• Design strategies:

  • Combining anti-CEACAM5 binding with immune effector cell engagement (T cells, NK cells)

  • Potential to simultaneously block inhibitory CEACAM5-CEACAM1 interactions while recruiting immune cells

3. CAR-T cell therapy:

• Engineered T-cell approaches:

  • Development of chimeric antigen receptors targeting CEACAM5-specific epitopes

  • Challenges include addressing on-target/off-tumor effects due to CEACAM5 expression in normal tissues

4. Radioimmunotherapy:

• Targeted radiation delivery:

  • Conjugation of anti-CEACAM5 antibodies with radioisotopes

  • Potential for treating microscopic disease and overcoming resistance mechanisms

5. Structure-guided antibody engineering:

• Affinity optimization:

  • Using high-resolution structural data of epitope-paratope interfaces

  • Rational design of improved binding properties

• Novel binding domains:

  • Development of alternative binding scaffolds (nanobodies, affibodies) targeting CEACAM5

These emerging applications leverage the specificity of anti-CEACAM5 antibodies while addressing the complex challenges of targeting tumor antigens that may also be expressed at lower levels in normal tissues.

How might advances in antibody engineering enhance the utility of anti-CEACAM5 recombinant antibodies?

Advanced antibody engineering approaches offer significant potential for enhancing anti-CEACAM5 antibodies:

1. Affinity maturation strategies:

• Directed evolution approaches:

  • Phage display with error-prone PCR to generate variant libraries

  • Selection under increasingly stringent conditions

• Computational design:

  • In silico modeling of antibody-antigen interactions based on structural data

  • Prediction of beneficial mutations to enhance binding

2. Format diversification:

• Fragment-based formats:

  • Single-chain variable fragments (scFvs)

  • Fab fragments for improved tissue penetration

• Multispecific designs:

  • Bispecific antibodies targeting CEACAM5 and immune checkpoint molecules

  • Dual-targeting of CEACAM5 and other tumor-associated antigens

3. Fc engineering:

• Enhanced effector functions:

  • Afucosylation to increase antibody-dependent cellular cytotoxicity

  • Point mutations to modulate complement activation

• Extended half-life:

  • Fc mutations that enhance FcRn binding

  • PEGylation or fusion to albumin-binding domains

4. Payload development for ADCs:

• Novel linker chemistry:

  • Stimulus-responsive linkers (pH, protease-sensitive)

  • Increasing drug-to-antibody ratio while maintaining stability

• Alternative payloads:

  • Exploring immunomodulatory agents beyond traditional cytotoxics

  • Site-specific conjugation approaches

5. Glycoengineering:

• Controlled glycan profiles:

  • Uniform glycosylation patterns for consistent function

  • Removal of immunogenic glycan structures

• Strategic glycan modification:

  • Addition of sialic acids to reduce immunogenicity

  • Afucosylation to enhance NK cell activation