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

Clin Cancer Res. 2003 Sep 1;9(10 Pt 2):3845S-53S.

Abstract

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 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Binding, Competitive
  • Cell Line, Tumor
  • Enzyme-Linked Immunosorbent Assay
  • Gene Library
  • Glycosylation
  • Humans
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Variable Region / chemistry
  • Immunohistochemistry
  • Kinetics
  • Mice
  • Mice, Inbred BALB C
  • Models, Molecular
  • Molecular Sequence Data
  • Mucin-1 / chemistry*
  • Neoplasms / therapy*
  • Peptide Library
  • Peptides / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid

Substances

  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Mucin-1
  • Peptide Library
  • Peptides