Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives.
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.
Schoonjans
R
R
Molecular Immunology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium.
Willems
A
A
Schoonooghe
S
S
Fiers
W
W
Grooten
J
J
Mertens
N
N
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
D000276:Adjuvants, Immunologic; D018033:Antibodies, Bispecific; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins