384©X.Tian et al antibody m396 as indicated by the complex crystal D95 ofm396-VL Concordantly,the elect as well as a MERS-CoV-spe action was also observed in the model of 2019-nCoV- antibody m336 developed by our laboratory [15],and RBD-m396, forming by R408 (RBD)and D95 that mos actions between antibody F26G19 or 8R 12] measured the binding kinetics using BLI.An irrelevant and the RBD in 2019-nCoV decreased significantly anti-CD40 antibody was used as a negative control due to the lack Similarly,the antibo ly m396,which .RBD-80 80-R162 ight bindi RBD (Figure 1(d)) only show of the goR-binding uc n the RBD of SARS CoV are not conserved on RBD of 2019-nCoV(Figure high-resolution structure of 2019-nCoV RBD and 1(c)),it is unlikely that the antibody 80R could effec understand why it could not be recognized by these tively recognize 2019-nCo t is urgent to antibodi SARS-C01 ctivity of ant hiCoanheipoha01pSeYoPoP was fo d to R rapid development of vaccines and therapeutic antibodies against 2019-nCov Koff of1.16× 10 s)binding kinetics. resulting in expressed ody was did no om blo (Figure 1(f) This ang e with th on of the to rcombinant S protein 5].Tofurther elucidate the binding epitopes of CR,we measured the compe and human ACE2 for the bin n)In both 20 120 COV RBD n biosensors 67loop form extensive contact,including at least human ACE2 in solution followed by the addition of seven pairs ofhydrogen bonds,with the receptor.Nota the test antibodies in the presence of ACE2.As bly,R426 on the forth a helix in SARS-CoV RBD builds shown in Figure 1(g),the antibody CR3022 did not 201-Co RBD Th the binding RBD)to on in 2019-nCov distinct from the SARs-CoV antibodies which recognizes an epitope that does not overlap with the ACE2 bind nt restingly,a lysine 019 largely from cement ues with D30 on ACE?which ver the hindin es in the ca hility to er ability.These data indicate that the RBD in S protein Some of the most poten Indeed,we r 0f2019- ACE2 ng anti ing o (BI found that 2019-nCov RBD hound potently to AcE2 ing that it is necessary to develop novel monoclonal The calculated affinity (K)of 2019-nCoV RBD with tibodies that could bind specifically to019-nCov human ACE2 was 15. 2nM (Figure 1()),which is com D Interestingly,it was reported that the antibody sp. pro wild-typ could be the ntial for the ne ml and the ndthattheexpressed019-nCoV RBD protein is func- be generated with CR3022 5).Furthermore,the mix tional [2]. ture of CR3022 and CR3014 neutralized SARS-CoVantibody m396 as indicated by the complex crystal structure [10] are invariably conserved in 2019-nCoV RBD (Figure 1(d)). In the structure of SARS-CoV￾RBD-m396, R395 in RBD formed a salt bridge with D95 of m396-VL. Concordantly, the electrostatic inter￾action was also observed in the model of 2019-nCoV￾RBD-m396, forming by R408 (RBD) and D95 (m396-VL). This analysis suggests that some SARS￾CoV-specific monoclonal antibodies may be effective in neutralizing 2019-nCoV. In contrast, the inter￾actions between antibody F26G19 [11] or 80R [12] and the RBD in 2019-nCoV decreased significantly due to the lack of salt bridges formed by R426-D56 in SARS-CoV-RBD-F26G19 or D480-R162 in SARS￾CoV-RBD-80R, respectively. Furthermore, while most of the 80R-binding residues on the RBD of SARS￾CoV are not conserved on RBD of 2019-nCoV (Figure 1(c)), it is unlikely that the antibody 80R could effec￾tively recognize 2019-nCoV. Therefore, it is urgent to experimentally determine the cross-reactivity of anti￾SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. In this study, we first expressed and purified 2019- nCoV RBD protein. We also predicted the confor￾mations of 2019-nCoV RBD and its complex with the putative receptor, human ACE2. Comparison of the interaction between the complex of ACE2 [13] and SARS-CoV RBD and homology model of ACE2 and 2019-nCoV RBD revealed similar binding modes (data not shown). In both complexes, β5–β6 loop and β6–β7 loop form extensive contact, including at least seven pairs of hydrogen bonds, with the receptor. Nota￾bly, R426 on the forth α helix in SARS-CoV RBD builds a salt bridge with E329 and a hydrogen bond with Q325 on ACE2. However, the arginine (R426 in SARS-CoV RBD) to asparagine (N439) mutation in 2019-nCoV RBD abolished the strong polar interactions, which may induce a decrease in the binding affinity between RBD and the receptor. Interestingly, a lysine (K417 in 2019-nCoV RBD) replacement of valine (V404 in SARS-CoV RBD) on β6 formed an extra salt bridge with D30 on ACE2, which may recover the binding ability. These data indicate that the RBD in S protein of 2019-nCoV may bind to ACE2 with a similar affinity as SARS-CoV RBD does. Indeed, we measured the binding of 2019-nCoV RBD to human ACE2 by the biolayer interferometry binding (BLI) assay, and found that 2019-nCoV RBD bound potently to ACE2. The calculated affinity (KD) of 2019-nCoV RBD with human ACE2 was 15.2 nM (Figure 1(f)), which is com￾parable to that of SARS-CoV spike protein with human ACE2 (15 nM) [14]. These results indicate that ACE2 could be the potential receptor for the new coronavirus, and that the expressed 2019-nCoV RBD protein is func￾tional [2]. Next, we expressed and purified several representa￾tive SARS-CoV-specific antibodies which have been reported to target RBD and possess potent neutralizing activities, including m396 [3], CR3014 [4], CR3022 [5], as well as a MERS-CoV-specific human monoclonal antibody m336 developed by our laboratory [15], and measured their binding ability to 2019-nCoV RBD by ELISA (Figure 1(e)). Surprisingly, we found that most of these antibodies did not show evident binding to 2019-nCoV RBD. To confirm this result, we further measured the binding kinetics using BLI. An irrelevant anti-CD40 antibody was used as a negative control. Similarly, the antibody m396, which was predicted to bind 2019-nCoV RBD (Figure 1(d)), only showed slight binding at the highest measured concentration (2.0 µM). Further studies are needed to solve the high-resolution structure of 2019-nCoV RBD and understand why it could not be recognized by these antibodies. Notably, one SARS-CoV-specific antibody, CR3022, was found to bind potently with 2019-nCoV RBD as determined by ELISA and BLI (Figure 1(e,f)). It fol￾lowed a fast-on (kon of 1.84 × 105 Ms−1 ) and slow-off (koff of 1.16 × 10−3 s −1 ) binding kinetics, resulting in a KD of 6.3 nM (Figure 1(f)). This antibody was iso￾lated from blood of a convalescent SARS patient and did not compete with the antibody CR3014 for binding to recombinant S protein [5]. To further elucidate the binding epitopes of CR3022, we measured the compe￾tition of CR3022 and human ACE2 for the binding to 2019-nCoV RBD. The streptavidin biosensors labelled with biotinylated 2019-nCoV RBD were saturated with human ACE2 in solution, followed by the addition of the test antibodies in the presence of ACE2. As shown in Figure 1(g), the antibody CR3022 did not show any competition with ACE2 for the binding to 2019-nCoV RBD. These results suggest that CR3022, distinct from the other two SARS-CoV antibodies, recognizes an epitope that does not overlap with the ACE2 binding site of 2019-nCoV RBD. The RBD of 2019-nCoV differs largely from the SARS-CoV at the C-terminus residues (Figure 1(c)). Our results implied that such a difference did not result in drastic changes in the capability to engage the ACE2 receptor, but had a critical impact on the cross-reactiv￾ity of neutralizing antibodies. Some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the receptor binding site of SARS￾CoV failed to bind 2019-nCoV spike protein, indicat￾ing that it is necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD. Interestingly, it was reported that the antibody CR3022 completely neutralized both the wild-type SARS-CoV and the CR3014 escape viruses at a concen￾tration of 23.5 μg/ml, and that no escape variants could be generated with CR3022 [5]. Furthermore, the mix￾ture of CR3022 and CR3014 neutralized SARS-CoV 384 X. Tian et al