key: cord-0708917-ycuiso0g
authors: Li, Wei; Drelich, Aleksandra; Martinez, David R.; Gralinski, Lisa; Chen, Chuan; Sun, Zehua; Liu, Xianglei; Zhelev, Doncho; Zhang, Liyong; Peterson, Eric C.; Conard, Alex; Mellors, John W.; Tseng, Chien-Te; Baric, Ralph S.; Dimitrov, Dimiter S.
title: Potent neutralization of SARS-CoV-2 in vitro and in an animal model by a human monoclonal antibody
date: 2020-05-14
journal: bioRxiv
DOI: 10.1101/2020.05.13.093088
sha: a036567a03d34c9633f3509906f0a3d84853a77c
doc_id: 708917
cord_uid: ycuiso0g

Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized live SARS-CoV-2 with exceptional potency as measured by two different assays. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 protected transgenic mice expressing hACE2 from high-titer intranasal SARS-CoV-2 challenge (105 plaque forming units). Another antibody, VH ab5 did not compete with hACE2 and ab1, and did not neutralize SARS-CoV-2 although its affinity was comparable to that of ab1. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

against emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV) (4) , Middle East respiratory syndrome coronavirus (MERS-CoV) (5) and henipaviruses (6, 7) , respectively, which are also highly effective in animal models of infection (8) (9) (10) (11) ; one of them was administered on a compassionate basis to humans exposed to henipaviruses and successfully evaluated in a clinical trial (12) .

Size and diversity of phage-displayed libraries are critical for rapid selection of high affinity antibodies without the need for additional affinity maturation. Our exceptionally potent antibody against the MERS-CoV, m336, was directly selected from very large (size ~10 11 clones) library from 50 individuals (5) . However, another potent antibody, m102.4, against henipavirusses was additionally affinity matured from its predecessor selected from smaller library (size ~10 10 clones) from 10 individuals (7, 13) . Thus, to generate high affinity and safe mAbs we constructed eight very large (size ~ 10 11 clones each) naive human antibody libraries in Fab, scFv or VH format using PBMCs from 490 individuals total obtained before the SARS-CoV-2 outbreak. Four of the libraries were based on single human VH domains where CDRs (except CDR1 which was mutagenized or grafted) from our other libraries were grafted as previously described (14) .

Another important factor to consider when selecting effective mAbs is the appropriate antigen. Similar to SARS-CoV, SARS-CoV-2 uses the spike glycoprotein (S) to enter into host cells. The S receptor binding domain (RBD) binds to its receptor, the human angiotensinconverting enzyme 2 (hACE2), thus initiating series of events leading to virus entry into cells (15, 16) . We have previously characterized the function of the SARS-CoV S glycoprotein and identified its RBD which is stable in isolation (17). The RBD was then used as an antigen to pan phage displayed antibody libraries; we identified potent antibodies (5, 8) more rapidly and the antibodies were more potent than when we used whole S protein or S2 (unpublished). In addition, the SARS-CoV RBD based immunogens are highly immunogenic and elicit neutralizing antibodies which protect against SARS-CoV infections (18). Thus, to identify SARS-CoV-2 mAbs, we generated two variants of the SARS-CoV-2 RBD (aa 330-532) (Fig.   S1 ) and used them as antigens for panning of our eight libraries.

Panels of high-affinity binders to RBD in Fab, scFv and VH domain formats were identified. There was no preferential use of any antibody VH gene (an example for a panel of binders selected from the scFv library is shown in Fig. S2A ) and the number of somatic mutations was relatively low (Fig. S2B , for the same panel of binders as in Fig. S2A ). The mAbs can be divided into two groups in terms of their competition with hACE2. Two representatives of each group are Fab ab1 and VH ab5. To further increase their binding through avidity effects and extend their half-live in vivo they were converted to IgG1 and VH-Fc fusion formats, respectively. Ab1 was characterized in more details because of its potential for prophylaxis and therapy of SARS-CoV-2 infection.

The Fab and IgG1 ab1 bound strongly to the RBD (Fig. 1A ) and the whole SARS-CoV-2 S1 protein ( Fig. 1B) as measured by ELISA. The Fab ab1 equilibrium dissociation constant, Kd, as measured by the biolayer interferometry technology (BLItz), was 1.5 nM (Fig. 1C) . The IgG1 ab1 bound with high (160 pM) avidity to recombinant RBD (Fig. 1D) . IgG1 ab1 bound cell surface associated native S glycoprotein suggesting that the conformation of its epitope on the RBD in isolation is close to that in the native S protein (Fig. 2, S3 ). The binding of IgG1 ab1 was of higher avidity than that of hACE2-Fc (Fig. 2B) . Binding of ab1 was specific for the SARS-CoV-2 RBD; it did not bind to the SARS-CoV S1 (Fig. 3A ) nor to cells that do not express SARS-CoV-2 S glycoprotein ( Fig. 2A ). Ab1 competed with hACE2 for binding to the RBD (Fig.   3B ) indicating possible neutralization of the virus by preventing its binding to the receptor. It did not compete with the IgG1 CR3022 (Fig. 3C) , which also binds to SARS-CoV (19) and with ab5 ( Fig. 3D ).

IgG1 ab1 exhibited potent neutralizing activity in two different assays using replicationcompetent SARS-CoV-2 -a microneutralization-based assay (100% neutralization, NT100, at <400 ng/ml) (Fig. 4A ) and a luciferase reporter gene assay (IC50 = 200 ng/ml) (Fig. 4B ). In agreement with the specificity of binding to the SARS-CoV-2 S1 and not to the SARS-CoV S1 the IgG1 ab1 did not neutralize live SARS-CoV (Fig. 4A ,C). The IgG1 m336 (5) control which is a potent neutralizer of MERS-CoV, did not exhibit any neutralizing activity against SARS-CoV-2 (Fig. 4) . The VH ab5 and VH-Fc ab5 bound the RBD with high affinity and avidity ( Fig.   S4A .B) but did not compete with hACE-2 ( (out of four) mice at the lower dose but at the higher dose all four mice were protected (9) . The in vivo protection also indicates that IgG1 ab1 can achieve neutralizing concentrations in the respiratory tract. This is the first report of in vivo activity of a human monoclonal antibody against SARS-CoV-2.

Interestingly, Fab ab1 had only several somatic mutations compared to the closest germline predecessor genes. This implies that ab1-like antibodies could be elicited relatively quickly by using RBD-based immunogens especially in some individuals with naïve mature B cells expressing the germline predecessors of ab1. This is in contrast to the highly mutated broadly neutralizing HIV-1 antibodies that require long maturation times, are difficult to elicit and their germline predecessors cannot bind native HIV-1 envelope glycoproteins (21, 22). The RBD of the MERS-CoV S protein was previously shown to elicit neutralizing antibodies (23, 24). For SARS-CoV-2 only a few somatic mutations would be sufficient to generate potent neutralizing antibodies against the SARS-CoV-2 RBD which is a major difference from the elicitation of broadly neutralizing antibodies against HIV-1 which requires complex maturation pathways (21, 25-28). The germline-like nature of the newly identified mAb ab1 also suggests that it has excellent developability properties that could accelerate its development for prophylaxis and therapy of SARS-CoV-2 infection (29).

To further assess the developability (drugability) of the antibodies their sequences were analyzed online (opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php); no obvious liabilities were found. In addition, we used dynamic light scattering (DLS) and size exclusion chromatography to evaluate its propensity for aggregation. IgG1 ab1 at a concentration of 2 mg/ml did not aggregate for six days incubation at 37°C as measured by DLS (Fig. 6A) ; there were no high molecular weight species in freshly prepared IgG1 ab1 also as measured by size exclusion chromatography (SEC) (Fig. 6B ). IgG1 ab1 also did not bind to the human cell line 293T ( Fig. 2A ) even at very high concentration (1 μM) which is about 660-fold higher than its Kd indicating absence of non-specific binding to many membrane-associated human proteins.

The IgG1 ab1 also did not bind to 5,300 human membrane-associated proteins as measured by a membrane proteome array (Fig. 7) .

The high affinity/avidity and specificity of IgG1 ab1 along with potent neutralization of virus and good developability properties suggests its potential use for prophylaxis and therapy of SARS-CoV-2 infection. Because it strongly competes with hACE2 indicating a certain degree of mimicry, one can speculate that mutations in the RBD may also lead to inefficient entry into cells and infection. However, in the unlikely case of mutations that decrease the ab1 binding to RBD it can be used in combination with other mAbs including those we identified or in bi(multi)specific formats to prevent infection of such SARS-CoV-2 isolates. Ab1 could also be used to select appropriate epitopes for vaccine immunogens and for diagnosis of SARS-CoV-2

infections. The identification of neutralizing mAbs within days of target availability shows the potential value of large antibody libraries for rapid response to emerging viruses. 

IgG1 CR3022 (30) and IgG1 S230 genes were synthesized by IDT (Coralville, Iowa). MERS-CoV-specific IgG1 m336 antibody was expressed in human mammalian cell as described previously (5) . The ACE2 gene was ordered from OriGene (Rockville, MD). The RBD domain (residues 330-532) and S1 domain (residues 14-675) and ACE2 (residues 18-740) genes were cloned into plasmid which carries a CMV promotor with an intron, human IgG1 Fc region and Woodchuck posttranscriptional regulatory element (WPRE) to generate the RBD-Fc, S1-Fc and ACE2-Fc expression plasmids. The RBD-avi-his protein with an avi tag followed by a 6×His tag at C-terminal was subcloned similarly. These proteins were expressed with Expi293 expression system (Thermo Fisher Scientific) and purified with protein A resin (GenScript) and by Ni-NTA resin (Thermo Fisher Scientific). The Fab CR3022 antibody gene with a His tag was cloned into pCAT2 plasmid (developed in house) for expression in HB2151 bacteria and purified with Ni-NTA resin. Protein purity was estimated as >95% by SDS-PAGE and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare). 

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