Ji Woong Kim a,1, Kyun Heo a,b,c,1, Hyun Jung Kim b, Youngki Yoo d, Hyun-Soo Cho d, Hui Jeong Jang e, Ho-Young Lee e, In Young Ko f, Ju Rang Woo f, Yea Bin Cho a, Ji Hyun Lee b, Ha Rim Yang b, Ha Gyeong Shin b, Hye Lim Choi b, Kyusang Hwang b, Sokho Kim g, Hanseong Kim h, Kwangrok Chun i, Sukmook Lee a,b,c
aDepartment of Biochemistry, Kookmin University, Seoul, 02707, Republic of Korea
bDepartment of Biopharmaceutical Chemistry, Kookmin University, Seoul, 02707, Republic of Korea
cAntibody Research Institute, Kookmin University, Seoul, 02707, Republic of Korea
dDepartment of Systems Biology, Yonsei University, Seoul, 03722, Republic of Korea
eDepartment of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul, 13620, Republic of Korea
fNew Drug Development Center, KBIOHealth, Cheongju, 28160, Republic of Korea
gResearch Center, KNOTUS Co. Ltd., Incheon, 22014, Republic of Korea
hBaobab AiBIO, POSCO Green Building, Yonsei University, Incheon, 21983, Republic of Korea
iR&D Center, Binex, Incheon, 21999, Republic of Korea
1These authors contributed equally to this work.
Corresponding author : Sukmook Lee
Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.