Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope
Manoj Chugh1, Vladimir Piskarev2, Oxana Galanina3
11 Agappe Diagnostics Ltd, Kochi, India.
Abstract:
A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLeA, as well as its defucosylated form sialyl LeC (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galβ1-3GlcNAcβ (LeC), thus resembling specificity of human natural cancer-associated anti-LeC antibodies. We propose that cancer-specific glycopeptide epitope includes Galβ1-3GlcNAcβ fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.
Insights
Monoclonal antibodies targeting cancer-associated glycoprotein CA19.9 unexpectedly recognized a different epitope. These antibodies bind to a common motif resembling natural anti-LeC antibodies, suggesting a novel cancer-specific epitope.
Area of Science:
- Biochemistry
- Immunology
- Glycobiology
Background:
- Cancer biomarkers are crucial for early detection and diagnosis.
- CA19.9 is a widely used tumor marker, but its precise epitope specificity remains debated.
- Monoclonal antibodies are key tools for developing diagnostic assays.
Purpose of the Study:
- To generate and characterize monoclonal antibodies against cancer-associated glycoprotein CA19.9.
- To elucidate the fine epitope specificity of these antibodies.
- To propose a novel cancer-specific glycopeptide epitope.
Main Methods:
- Immunization of mice with CA19.9 glycoprotein.
- Selection of optimal antibody pairs for sandwich assays.
- Epitope mapping using printed glycan arrays, ELISA, and inhibitory ELISA.
- Analysis of glycan-binding profiles and glycotopes.
Main Results:
- Two monoclonal antibodies were selected for CA19.9 detection.
- Unexpectedly, antibodies did not bind the canonical SiaLeA epitope or the CA-50 epitope.
- Antibodies recognized a common Galβ1-3GlcNAcβ (LeC) glycotrope, similar to natural anti-LeC antibodies.
Conclusions:
- The generated antibodies do not target the primary CA19.9 epitope.
- The antibodies' specificity suggests a potential role for the Galβ1-3GlcNAcβ motif in cancer recognition.
- A novel cancer-specific glycopeptide epitope, involving the Galβ1-3GlcNAcβ fragment and adjacent amino acids, is proposed.
