A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain.
Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrow's medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.
Schoonjans
R
R
Department of Molecular Biology, Molecular Immunology Unit, Flanders Interuniversity, Institute for Biotechnology (VIB), University of Ghent, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
Willems
A
A
Schoonooghe
S
S
Leoen
J
J
Grooten
J
J
Mertens
N
N
D000469:Alkaline Phosphatase / Q000276:immunology; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western; D002455:Cell Division; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009194:Myeloma Proteins / Q000235:genetics / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects; D014407:Tumor Cells, Cultured
D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D009194:Myeloma Proteins; D011993:Recombinant Fusion Proteins; C017551:myeloma immunoglobulins; D000469:Alkaline Phosphatase