A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing

• Published in: Journal of immunological methods 2020-08, Vol.483, p.112811-112811, Article 112811
• Main Authors: Huang, Shuyu, Segués, Aina, Hulsik, David Lutje, Zaiss, Dietmar M., Sijts, Alice J.A.M., van Duijnhoven, Sander M.J., van Elsas, Andrea
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bispecific - biosynthesis; Antibodies, Bispecific - genetics; Antibodies, Bispecific - immunology; Antibody chain association; Antibody Specificity; Bispecific antibody; Charge-pairs; CHO Cells; Controlled fab-arm exchange; Cricetulus; ErbB Receptors - genetics; ErbB Receptors - immunology; ErbB Receptors - metabolism; Glutamate Carboxypeptidase II - genetics; Glutamate Carboxypeptidase II - immunology; Glutamate Carboxypeptidase II - metabolism; Immunoglobulin Fab Fragments - biosynthesis; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Fc Fragments - biosynthesis; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - immunology; Immunoglobulin G - immunology; Knobs-into-holes; Membrane Glycoproteins - genetics; Membrane Glycoproteins - immunology; Membrane Glycoproteins - metabolism; Mice; Molecular Weight; Mutation; Proof of Concept Study; Protein Multimerization; Single-Domain Antibodies - biosynthesis; Single-Domain Antibodies - genetics; Single-Domain Antibodies - immunology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2020.112811

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity. •Bispecific antibodies with format Fab x sdAb-Fc constructed using cFAE, CP and KIH.•Bispecific antibodies with format Fab x sdAb-Fc avoid heavy-light chain mis-pairing.•Bispecific antibodies with format Fab x sdAb-Fc provide additional purification methods.