**1. Introduction**

Since the pandemic outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first discovered in Wuhan, China, in 2019, the rapidly growing number of infected people and casualties has posed a serious global threat [1]. SARS-CoV-2 causes severe respiratory symptoms that are accompanied by high fever, cough, and severe pneumonia [2], and although the mortality rate has been reported to be lower than that of severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV), the overall risk remains highly significant, and thus novel prophylactic agents such as therapeutic drugs and vaccines are urgently in need.

Among the four coronavirus genera (α, β, γ, and δ), SARS-CoV-2 belongs to the β-coronaviruses and is an enveloped, positive-sense single-stranded RNA (or (+) ssRNA) virus, the RNA genome of which encodes structural proteins including the spike (S)

**Citation:** Kim, Y.J.; Lee, M.H.; Lee, S.-R.; Chung, H.-Y.; Kim, K.; Lee, T.G.; Kim, D.Y. Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library. *Int. J. Mol. Sci.* **2021**, *22*, 1913. https://doi.org/ 10.3390/ijms22041913

Academic Editor: Philippe Rondard Received: 18 January 2021 Accepted: 12 February 2021 Published: 15 February 2021

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protein [3,4]. SARS-CoV-2 shares similarities in its genome sequences with those of SARS-CoV and MERS-CoV, which are respectively about 79.5% and 50% similar, indicating homologous structures and similar infectious pathways to SARS-CoV [5].

As in all coronaviruses, the S protein is present on the surface of the virus and plays a critical role in the viral entry to host cells [6,7]. The S protein consists of two subunits, S1 and S2, which are non-covalently associated as a homotrimeric form that comprises a prefusion state. The receptor-binding domain (RBD, residues 387–516) of the S1 subunit consists of a core domain and a receptor-binding motif (RBM, residues 438–505), and this motif directly engages with the host receptor, known as angiotensin-converting enzyme 2 (ACE2) [8]. Upon entry of the virus into cells, the RBD of the S1 subunit recognizes ACE2 on the surface of host cells as a receptor, while the S2 subunit has a role in viral fusion with host cell membranes and is primed by the S protein cleavage at the S1/S2 and S2 sites on the S2 subunit through intracellular proteases such as TMPRSS2, triggering the conformational change of the S protein to the postfusion state [8–13].

Due to the urgent situation in which no drugs or vaccines are available, researchers and medical doctors have worked in close cooperation to develop a variety of therapeutic approaches, mostly repurposed from existing drugs, including nucleoside analogs such as remdesivir [14–16], antiparasitics such as chloroquine [17], protease inhibitors such as lopinavir and ritonavir [18], indole-derivate molecules such as arbidol [19], plasma therapy from convalescent patients who recovered from the infection [20], and, lastly, antibodies that treat the viral infection by blocking the S protein or pro-inflammatory cytokines, such as IL-6, TNF-α, GM-CSF, and IFN-γ [21,22].

Monoclonal antibodies (mAbs) have been recognized as significant biologics in therapeutic fields and are now rapidly taking a position as an alternative treatment that complements vaccines in working against newly emerging pathogenic viruses, such as SARS-CoV-2 [22–25]. Since viral neutralization by targeting the S protein has previously been shown to correlate with therapeutic efficacy in animal models [26], tremendous efforts have been made, based on the structural information of the S protein and its critical role in viral entry, to discover neutralizing mAbs that block the RBD of the S1 subunit through a variety of approaches, such as phage display library selection [27–30]; antibody selection through immunization of animals, such as humanized mice, dromedary camels, or sharks [31–34]; and antibody isolation from memory or plasma B cells of naturally infected human donors [31,35–39]. At the time of writing, 198 antibodies programs are in discovery and development phases globally, among which 66 are going through clinical trials (phase 1/2/3). In particular, 122 antibodies programs (~62%) are known to target the S protein of SARS-CoV-2, highlighting the importance of the S protein as a target. Last November, the United States Food and Drug Administration (US FDA) approved two antibodies targeting the S protein (REGN-COV2 (REGN10933 + REGN10987) and Bamlanivimab from Regeneron and Eli Lilly, respectively) for the treatment of COVID-19 patients [21].

However, the mAb approach, mainly based on the immunoglobulin G (IgG) format, has a drawback in that it relies on mammalian cell lines for the production of antibodies, which is costly and time-consuming. Moreover, viruses can easily evolve to generate RBD variants with mutations avoiding immune responses [39]. Therefore, in order to protect against the immune escapers, it would be useful to identify a selection of mAbs broadly acting on different epitopes that could contribute to a therapeutic antibody cocktail that might induce resistance to mutations in the RBD variants [29,40,41].

In this study, we panned a human Fab synthetic phage display library on the RBD of the SARS-CoV-2 S protein and obtained human mAbs with desirable properties that successfully neutralized the viral entry upon SARS-CoV-2 infection. We anticipate that these mAbs can be further developed as a promising antibody therapy against the pathogenic virus and as tools for diagnosis.
