Purpose: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells. Experimental Design: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 × 105 calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA. Results: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K a) range between 8 × 107 and 109 M -1. Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23). Conclusions: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.
|Original language||English (US)|
|Journal||Clinical Cancer Research|
|Issue number||10 II|
|State||Published - Oct 1 2003|
ASJC Scopus subject areas
- Cancer Research