Humanization and epitope mapping of the H23 anti-MUC1 monoclonal antibody reveals a dual epitope specificity.
The tumor-associated antigen MUC1 is a cell surface mucin that is expressed on the apical surface of most glandular epithelial cells, including the ducts of the breast, ovary, pancrease, lung and colon. During malignancy, epithelial tissues regularly display elevated levels of MUC1 in a non-polar fashion and in an underglycosylated form, exposing cryptic peptide and carbohydrate epitopes. As such, MUC1 is regarded a potential target for immunotherapeutical intervention. Murine monoclonal H23 antibody specifically recognizes a MUC1 epitope on the surface of human breast cancer cells. We describe the cloning of the variable domains of H23 and their expression in (Escherichia coli) E. coli as maltose-binding protein-scFv (MBP-scFv) fusions. We humanized H23 and evaluated the binding properties of the murine and the humanized recombinant forms, which were similar in affinity and specificity, but lower in apparent affinity in comparison to the original monoclonal IgG. We mapped the epitope of humanized H23 by affinity-selecting a phage-displayed random peptide library on humanized H23 scFv-displaying bacteria. Our results show that humanized H23 binds an epitope corresponding to the MUC1 tandem repeat and an additional epitope not related to MUC1. These epitopes are competitive, bound with similar affinities and are recognized by the original murine H23 monoclonal antibody as well.
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel.
Yariv
Y
Mazor
Iafa
I
Keydar
Itai
I
Benhar
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000276:immunology; D002352:Carrier Proteins; D003001:Cloning, Molecular; D018604:Epitope Mapping; D006801:Humans; D007135:Immunoglobulin Variable Region; D058619:Maltose-Binding Proteins; D051379:Mice; D008969:Molecular Sequence Data; D018396:Mucin-1; D009077:Mucins; D019151:Peptide Library; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D002352:Carrier Proteins; D007135:Immunoglobulin Variable Region; C434325:MUC-1 monoclonal antibody; C060192:MUC1 protein, human; D058619:Maltose-Binding Proteins; D018396:Mucin-1; D009077:Mucins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins