*Article* **Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library**

**Yu Jung Kim, Min Ho Lee, Se-Ra Lee, Hyo-Young Chung, Kwangmin Kim, Tae Gyu Lee and Dae Young Kim \*,†**

New Drug Development Center, Osong Medical Innovation Foundation, Cheongju-si, Chungcheongbuk-do 28160, Korea; yjkim@kbiohealth.kr (Y.J.K.); hpimh3@kbiohealth.kr (M.H.L.); srlee@kbiohealth.kr (S.-R.L.); hchung@kbiohealth.kr (H.-Y.C.); kwangmin.kim@kbiohealth.kr (K.K.); tglee17@gmail.com (T.G.L.)

**\*** Correspondence: kdypsh99@gmail.com; Tel.: +82-10-2460-4630

† Current address: PnP Biopharm Ltd., #1304, Acetechno Tower 8 cha, 11, Digital-ro 33 gil, Guro-gu, Seoul 08380, Korea.

**Abstract:** Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (*EC*<sup>50</sup> = 19–663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs' activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (*K*<sup>D</sup> = 0.08–1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.

**Keywords:** SARS-CoV-2; spike protein; receptor-binding domain; phage display; monoclonal antibody
