title: Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives.
abstract: Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
authors: Molecular Immunology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium.
authors: R
authors: R
authors: Schoonjans
authors: A
authors: A
authors: Willems
authors: S
authors: S
authors: Schoonooghe
authors: W
authors: W
authors: Fiers
authors: J
authors: J
authors: Grooten
authors: N
authors: N
authors: Mertens
mesh_terms: D000276:Adjuvants, Immunologic / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D001666:Binding Sites, Antibody / Q000235:genetics; D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D019281:Dimerization; D004355:Drug Stability; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000276:Adjuvants, Immunologic; D018033:Antibodies, Bispecific; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins