title: PH1-derived bivalent bibodies and trivalent tribodies bind differentially to shed and tumour cell-associated MUC1.
abstract: 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 (K(D) = 30 and 5 nM) of the PH1 bi- and tribodies compared with the parental Fab (K(D) = 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.
authors.affiliation: Molecular Immunology Laboratory, Department of Molecular Biology, Ghent University, Technologiepark 927, B-9052 Zwijnaarde, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Schoonooghe
authors.forename: Ingrid
authors.initials: I
authors.lastname: Burvenich
authors.forename: Liesbet
authors.initials: L
authors.lastname: Vervoort
authors.forename: Filip
authors.initials: F
authors.lastname: De Vos
authors.forename: Nico
authors.initials: N
authors.lastname: Mertens
authors.forename: Johan
authors.initials: J
authors.lastname: Grooten
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004705:Endocytosis; D005260:Female; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018396:Mucin-1 / Q000737:chemistry / Q000378:metabolism*; D009369:Neoplasms / Q000276:immunology; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D055767:Immobilized Proteins; D007140:Immunoglobulin Fab Fragments; C060192:MUC1 protein, human; D018396:Mucin-1