PH1-derived bivalent bibodies and trivalent tribodies bind differentially to shed and tumour cell-associated MUC1

• Published in: Protein engineering, design and selection design and selection, 2010-09, Vol.23 (9), p.721-728
• Main Authors: Schoonooghe, Steve, Burvenich, Ingrid, Vervoort, Liesbet, De Vos, Filip, Mertens, Nico, Grooten, Johan
• Format: Article
• Language: English
• Subjects: Animals; antibodies; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - metabolism; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antibody Affinity; antibody derivatives; Binding Sites, Antibody; bivalent; Cell Line, Tumor; Cloning, Molecular; Endocytosis; Female; Flow Cytometry; HEK293 Cells; Humans; Immobilized Proteins; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - metabolism; Mice; MUC1; Mucin-1 - chemistry; Mucin-1 - metabolism; Neoplasms - immunology; Protein Binding; Protein Engineering - methods; Protein Stability; trivalent
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzq044

Most adenocarcinomas express altered MUC1 as a tumour-associated antigen. Due to suboptimal glycosylation in tumour-associated MUC1, the apomucin core is exposed, revealing new epitopes for antibody-directed immunotherapy. The human PH1 Fab binds specifically to this MUC1 apomucin. We describe the engineering and functional characterization of bi- and trivalent recombinant antibody derivatives from the PH1 Fab. Bi- and tribodies were made using the disulfide-stabilized Fab fragment as a heterodimerization scaffold with PH1 single-chain variable fragments fused to either one or both Fab-chain C-termini. Immunoassays revealed 27- and 165-fold improved dissociation constants (KD = 30 and 5 nM) of the PH1 bi- and tribodies compared with the parental Fab (KD = 820 nM). Unexpectedly, major differences were seen in the ability of the antibody constructs to bind shed and tumour cell-tethered MUC1. While the tribody did not discriminate between both MUC1 forms, the bibody demonstrated preferential interaction with membrane-bound MUC1 compared with shed MUC1. This preferential recognition of membrane-bound MUC1, along with the high serum stability of the bibody, its intermediate size and efficient internalization by MUC1+ cells, makes the human PH1-derived bibody a valuable candidate as a cancer-targeting therapeutic.