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.