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Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin
Nor Adzimah Johdi1, Ruth Harman, Irene Sanjuan
1Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom.
Abstract:
Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50 nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.
Insights
Researchers developed a humanized antibody fragment (scFv) targeting tumour-associated fibronectin. This stable, tumor-localizing HuBC-1 scFv shows promise for developing novel cancer biopharmaceuticals.
Area of Science:
- Biotechnology
- Oncology
- Immunology
Background:
- Tumour-specific splice variants of fibronectin are key targets for cancer therapeutics.
- Murine BC-1 antibody recognizes a cryptic epitope on the EDB-FN splice variant.
- Initial antibody fragments (IgG) showed instability, necessitating humanization.
Purpose of the Study:
- To create a stable, humanized single-chain variable fragment (scFv) based on the BC-1 antibody.
- To assess the binding affinity, stability, and tumor-targeting capability of the engineered scFv.
Main Methods:
- Humanization of the BC-1 antibody variable domains.
- Construction and expression of the humanized scFv (HuBC-1 scFv) in E. coli.
- Characterization of epitope recognition, binding affinity (ELISA, SPR, Flow Cytometry), thermostability, and serum stability.
- Evaluation of tumor localization in human tumor xenograft models in mice.
Main Results:
- Successfully expressed HuBC-1 scFv in E. coli.
- HuBC-1 scFv retained conformationally-sensitive epitope recognition.
- Demonstrated good target affinity (approx. 50 nM) and stability (thermo- and serum-stable).
- Showed significant localization to human tumors in mouse xenograft models.
Conclusions:
- The HuBC-1 scFv is a stable and effective tumor-targeting agent.
- This engineered antibody fragment holds potential as a basis for future tumor-specific biopharmaceuticals.
