Inhibitory Effect of Human Anti-CA I Autoantibodies and Development of Monoclonal Antibody mAb 2B8 Targeting Carbonic
Petra Chalova1,2, Barbora Jankovicova3, Veronika Dvorakova3
1Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia.
Abstract:
Spontaneous tumor regression is a recognized phenomenon across various cancer types. Recent research emphasizes the alterations in autoantibodies against carbonic anhydrase I (CA I) (anti-CA I) levels as potential prognostic markers for various malignancies. Particularly, autoantibodies targeting CA I and II appear to induce cellular damage by inhibiting their respective protein's catalytic functions. Our study illuminates the profound impact of anti-CA I autoantibodies from patient serum on the esterase activity of human CA I, exhibiting inhibitory effects akin to the acetazolamide inhibitor. Concurrently, our newly synthesized mouse monoclonal IgG antibody, mAb 2B8, against human CA I showcased a potent inhibitory action. An in-depth exploration into mAb 2B8's binding dynamics with its target enzyme was undertaken. Leveraging epitope extraction and phage display library techniques, we identified the amino acid sequence DFWTYP (positions 191-196 of CA I) as crucial for mAb 2B8's interaction. In 3-D structural analysis, this sequence is spatially adjacent to a previously identified epitope (DFWTYP) that interacts with patient-derived autoantibodies. Critically, mAb 2B8 demonstrated an ability to infiltrate eukaryotic cells, engaging specifically with its intracytoplasmic target. This positions mAb 2B8 as a promising model for future studies aimed at tumor cell eradication.
Insights
Autoantibodies against carbonic anhydrase I (CA I) show promise as cancer markers. A new antibody, mAb 2B8, effectively inhibits CA I activity and targets it within cells, suggesting potential for tumor eradication.
Area of Science:
- Biochemistry
- Immunology
- Oncology
Background:
- Spontaneous tumor regression is observed in various cancers.
- Autoantibodies against carbonic anhydrase I (CA I) are emerging as potential prognostic markers for malignancies.
- Autoantibodies targeting CA I and II may induce cellular damage by inhibiting enzyme function.
Purpose of the Study:
- To investigate the impact of patient serum anti-CA I autoantibodies on human CA I esterase activity.
- To characterize a novel mouse monoclonal IgG antibody (mAb 2B8) against human CA I.
- To identify the binding epitope of mAb 2B8 and assess its cellular localization and target engagement.
Main Methods:
- Assay of human CA I esterase activity with patient serum and mAb 2B8.
- Epitope extraction and phage display library techniques for antibody-target interaction analysis.
- 3-D structural analysis and cellular infiltration studies of mAb 2B8.
Main Results:
- Patient serum anti-CA I autoantibodies inhibit human CA I esterase activity, similar to acetazolamide.
- The synthesized mAb 2B8 also potently inhibits CA I activity.
- mAb 2B8 binds to the DFWTYP sequence (CA I positions 191-196), which is spatially close to a patient autoantibody epitope.
- mAb 2B8 infiltrates eukaryotic cells and targets intracytoplasmic CA I.
Conclusions:
- Anti-CA I autoantibodies exhibit significant inhibitory effects on CA I enzymatic function.
- mAb 2B8 represents a specific and potent inhibitor of CA I with intracellular targeting capabilities.
- mAb 2B8 serves as a valuable model for developing strategies for tumor cell eradication.
