Selection and characterization of anti-MUC-1 scFvs intended for targeted therapy.
OBJECTIVE
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.


METHODS
A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) 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 x 10(7) and 10(9) 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.
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006031:Glycosylation; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry*; D009369:Neoplasms / Q000628:therapy*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018396:Mucin-1; D019151:Peptide Library; D010455:Peptides