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The rapid emergence of Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome 2 coronavirus (SARS-CoV-2) as a pandemic that presents an urgent human health crisis. Many SARS-CoV-2 neutralizing antibodies (NAbs) were developed with efﬁcient therapeutic potential. NAbs-based therapeutics against SARS-CoV-2 are being expedited to preclinical and clinical studies with two antibody drugs, LY3819253 (LY-CoV555) and REGN-COV2 (REGN10933 and REGN10987), approved by the US Food and Drug Administration for emergency use authorization for treating COVID-19. In this review, we provide a systemic overview of SARS-CoV-2 speciﬁc or cross-reactive NAbs and discuss their structures, functions and neutralization mechanisms. We provide insight into how these NAbs speciﬁc recognize the spike protein of SARS-CoV-2 or cross-react to other CoVs. We also summarize the challenges of NAbs therapeutics such as antibody-dependent enhancement and viral escape mutations. Such evidence is urgently needed to the development of antibody therapeutic interventions that are likely required to reduce the global burden of COVID-19. Statement of Signiﬁcance: The development of SARS-CoV-2 neutralizing antibodies (NAbs) has showed efﬁcacy for the treatment of COVID-19. We discuss in this review the current understanding of NAbs for their structures, functions and neutralization mechanisms, and the potential of cocktail NAb therapy against COVID-19. KEYWORDS: SARS-CoV-2; COVID-19; spike protein; neutralizing antibodies; hACE2 INTRODUCTION (RBD). However, there is currently no approved vaccines The rapid emergence of Coronavirus disease-2019 (COVID- or specific therapeutics against COVID-19. Considering 19) caused by severe acute respiratory syndrome 2 coron- the long term of clinical trials and the uncertainty of avirus (SARS-CoV-2) as a pandemic that has led to over 43 vaccine efficacy in human, the development of SARS-CoV- 2 NAbs with desired efficacy and safety profile is also a million cases and over 1 150 000 deaths worldwide . Many critical part of the strategy for the treatment of COVID-19. vaccines are currently under clinical or preclinical studies We provide here a systemic overview of the development [2, 3]. Most of the developing SARS-CoV-2 vaccines target of SARS-CoV-2 specific or cross-reactive NAbs, and the trimeric spike (S) glycoproteins and have the capacity discuss their structures, functions and neutralization to elicit high level of neutralizing antibodies (NAbs) mechanisms. that targeting S protein or its receptor binding domain To whom correspondence should be addressed. Qingbing Zheng. Email: email@example.com These authors contributed equally to this article. © The Author(s) 2020. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For Permissions, please email: jour- firstname.lastname@example.org 286 Antibody Therapeutics, 2020 Figure 1. Structures of SARS-CoV-2 S trimer and its receptor-binding domain (RBD). (A) Structural diagram of SARS-CoV-2 S trimers (PDB: 6VSB) with one RBD in complex with hACE2 (PDB: 6ACG). The trimeric protein of SARS-CoV-2 is shown in molecular surface with the ‘up’ RBD, ‘down’ RBD, NTD, SD1 and SD2 colored in red, blue, deep blue, misty rose and hot pink, respectively. The model of hACE2 is represented in gold. (B) A molecular surface representation of RBD (PDB: 7C01) with the hACE2 binding sites rendered in dark grey. (C) Cartoon representation of RBD with numbered alpha-helices and beta-sheets colored in red and blue, respectively. SARS-COV-2 SPIKE PROTEIN AND RECEPTOR and insertion into the host-cell membrane. Two heptad repeats (HR) including HR1 and HR2 associated from each The transmembrane S glycoprotein of coronaviruses protomer form a six-helix bundle that brings together the (CoVs) which serves as the machinery to fuse the viral and viral and host cellular membranes [12, 22, 29]. host cell membranes  is the primary immunogenic target for virus neutralization and vaccine design. The trimeric S protein is a type I fusion protein that comprises two SARS-COV-2 NAbs: STRUCTURES, FUNCTIONS functional subunits, with S1 (N-terminal) responsible for AND MECHENISMS mediating attachment to host cells and S2 (C-terminal) The clinical spectrum of the outcome of COVID-19 is for membrane fusion [5, 6]. Two subunits remain nonco- highly variable from mild flu-like symptoms to severe pneu- valently bound after proteolytic cleavage of the S protein monia . NAbs play an important role in virus clearance [7–14]. The S1 subunit contains a RBD and an N-terminal for patients with COVID-19. Vaccine induced NAbs in domain (NTD) and the former exhibits two discrete healthy individuals are important for the prevention of conformational states, including the closed ‘down’ state COVID-19, which is also a significant indicator for vaccine that shield the receptor binding regions, and the ‘up’ state, efficacy [31–33]. In recent months, many SARS-CoV-2- which, in the case of SARS-CoV and SARS-CoV-2, can derived NAbs have been emerging reported with excel- recognize the human angiotensin-converting enzyme 2 lent neutralizing and treatment potential, which promote (hACE2) receptor on the host cell [5, 6, 15–17] (Fig. 1). The the application of NAbs-based immune-therapy for the NTD may recognize sugar moieties upon initial attachment treatment of COVID-19. and might play a significant role in the transition of At the early beginning of the pandemic, the information S protein from prefusion to postfusion in some CoVs about the immune responses elicited in COVID-19 patients [18–21]. It can also provide immunogenic epitopes for has been collected, which provides us not only the prelimi- antibody targeting as indicated by that of MERS-CoV S nary understanding of virus induced host response, but also protein . The S2 subunit contains a fusion peptide near the possibility to screening human NAbs against SARS- which an additional protease cleavage site, refer to as S2’, CoV-2. A study on antibody responses in 30 COVID- locates . 19 patients from Chongqing, China indicated that NAbs The reported cryogenic electron microscopy (cryo-EM) against SARS-CoV-2 were detectable at the early stage or crystal structures of ectodomain of SARS-CoV-2 S pro- of COVID-19, peaked around 4–5 weeks, and gradually tein presented the high similarity to that of SARS-CoV, decreased within 3 months after the onset of symptoms as well as high flexibility of RBDs in either the ‘down’ or . Furthermore, NAb titer among intensive care unit ‘up’ states that similar to other CoVs [11, 15]. When an (ICU) patients were apparently higher and peaked earlier RBD stochastically swings upwards, the binding of hACE2 than that of non-ICU patients . Two independent to RBD can lock the RBD in the ‘up’ conformation and studies showed that NAbs titers in COVID-19 patients were trigger S1 dissociation, further drive a large irreversible strongly correlated with the concentration of anti-RBD conformational arrangement of S2 [6, 8, 9, 12–14, 23– IgGs, which indicate the RBD may contain the dominant 25]. During this change, the S2’ cleavage site is cleaved by neutralizing epitopes on the spike protein [36, 37]. Series of host proteases (e.g. TMPRSS2 for SARS-CoV and SARS- subsequent studies on the screening and characterization CoV-2 [26–28]) followed by the fusion peptide exposure Antibody Therapeutics, 2020 287 of SARS-CoV-2 NAbs confirmed that RBD is the target of Famous monoclonal antibody (mAb) CR3022 as repre- the most efficient NAbs (Table 1). sentatively, the binding of Type-II NAbs to spike protein would be sterically hindered unless at least two RBDs are in the ‘up’ state (Fig. 2B) [47, 48]. In some cases, the con- ditions for the binding may be harsher, requiring a certain NAbs TARGETING SARS-CoV-2 RBD deflection of the RBD to avoid the collision between Fab and S protein [48, 49]. Therefore, the epitopes are inacces- The structures of the S protein or its subunit RBD in sible and more hidden comparing to those of Type-I, which complex with NAbs have also been determined, which may account for the relatively less frequent report for this revealed the existence of several regions on RBD that can type of antibodies. Three NAbs including CR3022, EY6A stimulate immune system to elicit a considerable antibody and a single domain antibody VHH-72 that belong to Type- response to SARS-CoV-2. Furthermore, antibodies against II were reported. All of them bind to spike protein by lean- different epitopes of RBD may lead to the neutralization of ing on the bottom of RBD, with the interaction interface virus through different mechanisms. Here, we categorized distal from the receptor-binding site and mainly comprising these NAbs into at least four types (Type-I to Type-IV) β2 strand, α2 helices, α3 helices and the loops between (Table 1), pursuant to the conformations of bound RBD on them. Significantly, the binding sites of Type-II NAbs are S trimer and therefore lead to four distinct conformations highly conserved between SARS-CoV and SARS-CoV-2. of S trimers (All ‘down’ RBDs; one ‘up’ RBD; two ‘up’ For example, 24 out of 28 residues in the epitope of CR3022 RBDs and three ‘up’ RBDs) Interestingly, those NAbs that are conserved between two viruses, which enable the cross appear in the same type seem to prevent the viral infection binding of NAbs to these two viruses [48, 50]. with a similar mechanism. CR3022, which was isolated from a SARS patient, was The NAbs in Type-I can only bind to the ‘up’ RBD since initially verified to neutralize SARS-CoV but not SARS- the epitopes are sheltered or partially sheltered when RBD CoV-2 . However, a recent research investigated that was in the down state (Fig. 2A). The binding region that CR3022 can neutralize SARS-CoV-2 in a plaque-reduction Type-I NAbs target to is the flat surface on one side of neutralization assay . The binding of CR3022 could the top saddle-like surface of RBD, which comprised three promote the release of hACE2 from RBD and further short alpha-helices (α4, α5 and α7), two beta-strands (β5 reduce the stability of the prefusion state of spike protein and β6) and part of a flexible ridge-like loop (aa. 474–488) through locking RBD in the up state, which may present [38–44]. Epitopes of Type-I NAbs are extensively overlap- an uncommon neutralization mechanism . Similarly, ping with the binding site of hACE2, and further super- EY6A trapped essentially the RBDs in the ‘up’ state with imposition of the complex structures of NAbs-RBD and an extra rotation outwards by ∼ 25 , and can also make hACE2-RBD exhibited obvious steric hindrance or direct the premature prefusion-to-postfusion transition of spike binding-sites competition between NAbs and hACE2. The protein . In contrast, nanobody VHH-72, which was analysis implies that the neutralization mechanism of Type- isolated from a llama immunized with both SARS-CoV I NAbs depends on blocking the binding of hACE2 to spike protein and MERS-CoV spike protein, can easily SARS-CoV-2 RBD. Interestingly, most of NAbs (C105, engage the ‘up’ RBD without extra rotation of RBD due CV30, B38, CC12.1 and CC12.3) share the same germline to its small size . The peculiar binding characters of of heavy chain V-genes (IGHV3–53) [39, 41, 43–45], and the structures of the NAb-RBD complexes show that these VHH-72 to RBD, with an 834 Å of contact area in the nAbs attach to RBD with almost identical pose (Fig. 2A). vertical direction of RBD, confer VHH-72 two different Yuan et al.  have revealed the key motifs of neutralizing mechanism . The VHH-72 can disrupt the the IGHV3–53 germline-derived NAbs for binding to RBD dynamics and gives rise to the anticipatory triggering RBD, which include the NY motif from heavy-chain of S protein by trapping the ‘up’ configuration just like 32 33 complementarity-determining region 1 (HCDR1) and the how CR3022 and EY6A work . On the other hand, the SGGS motif from HCDR2. This information may distal framework that opposite to VHH-72 CDRs would 53 56 crash with the N-glycan occupancy at N322 as well as the help to investigate the specific binding mode of IGHV3-53 segment (aa 300–324) of hACE2, which means that VHH- germline-derived antibodies and the corresponding epi- 72 could also neutralize the virus by directly interfering topes. All NAbs exhibit exquisite potency in neutralizing with the hACE2 binding . In brief, the neutralization SARS-CoV-2 and have promising therapeutic effect. For mechanisms of Type-II NAbs against SARS-CoV-2 can be example, CC12.1 have an IC value of 0.019 µg/mL against pseudovirus in vitro . Administering a single attributed to the interference of the receptor-binding, or to 25 mg/kg dose of NAb B38 at 12 hours after viral triggering the premature transition of S protein from prefu- challenge could protect hACE2 transgenic mice against sion to postfusion by functionally mimicking the receptor SARS-CoV-2 infection with viral RNA copies in the binding and further trapping the RBD in the unstable ‘up’ lung significantly declined . For CB6, a single dose conformation [52–55].. of 50 mg/kg LALA mutant antibody before viral chal- Contrary to the Type-I NAbs, antibodies from Type-III, lenge could prophylactically prevent the rhesus macaque such as NAbs Fab 2–4, Fab 2–43 and BD23, can only bind from SARS-CoV-2 infection . These results indicated to the ‘down’ conformation of RBDs (Fig. 2C) [50, 56]. All that the blocking of hACE2 binding, either by direct of them would attach to the saddle-like surface of closed binding site competition or by steric hindrance, is an RBD from the top direction. As the fact that the dynamic effective strategy for antibody-mediated neutralization of conformational rearrangement of RBD is not accompanied SARS-CoV-2. by internal conformational changes of RBD, NAbs that 288 Antibody Therapeutics, 2020 Table 1. NAbs targeting SARS-CoV-2 RBD Type Antibody Antibody type Origin PDB Epitopes Neutralizing Cross- Protective efficacy Ref Name ID mechanism neutralizing activity I C105 Human IgG COVID-19- 6XCN, R403, D405, R408, Block no Neutralizing [39, 93] convalescent 6XCM T415-K417, D420-Y421, hACE2-RBD SARS-CoV-2 patient Y453, L455-N460, Y473, interaction pseudovirus with A475-G476, F486-N487, IC value of G502, Y505 26.1 ng/mL REGN10933 Recombinant Humanized mice 6XDG R403, K417, Y421, Y453, Block no Neutralizing  full-human and COVID-19- L455-F456, A475-G476, hACE2-RBD SARS-CoV-2 live antibodies convalescent E484-Y489, Q493 interaction, virus with IC value patients ADCC & ADCP of 37.4 pM CB6 Human IgG COVID-19- 7C01 R403, D405-E406, Block no A single dose of  convalescent R408-Q409, T415-K417, hACE2-RBD CB6-LALA patient D420-Y421, L455-N460, interaction (50 mg/kg) protected Y473-S477, F486-N487, the animal from Y489, Q493, Y495, SARS-CoV-2 N501-G502, G504-Y505 infection. B38 Human IgG COVID-19- 7BZ5 R403, D405-E406, Q409, Block no A single dose of B38  convalescent T415-K417, D420-Y421, hACE2-RBD (25 mg/kg) B38 patient Y452, L454-N460, interaction protected the hACE2 Y473-S477, F486-N487, transgenic mice from Y489-F490, Q493-G496, SARS-CoV-2 Q498, T500-V503, Y505 infection. CV30 Human IgG Infected 6XE1 R403, T415-K417, Block no Neutralizing  COVID-19 D420-Y421, Y453, hACE2-RBD SARS-CoV-2 live patients L455-N460, Y473-S477, interaction virus with IC value F486-N487, Y489, Q493, of 0.03 µg/mL T500, G502, Y505 CC12.3 Human IgG COVID-19- 6XC7 R403, D405, T415-K417, Block no Neutralizing [44, 45] convalescent D420-Y421, Y453, hACE2-RBD SARS-CoV-2 patient L455-N460, Y473-S477, interaction pseudovirus with F486-N487, Y489, Q493, IC value of G496, N501, Y505 0.018 µg/mL CC12.1 Human IgG COVID-19- 6XC3 R403, D405-E406, Block no Neutralizing convalescent R408-Q409, T415-K417, hACE2-RBD SARS-CoV-2 patient D420-Y421, Y453, interaction pseudovirus with L455-N460, Y473-S477, IC value of F486-N487, Y489, 0.019 µg/mL Q493-G496, Q498, T500-V503, Y505 Continued Antibody Therapeutics, 2020 289 Table 1. Continued Type Antibody Antibody type Origin PDB Epitopes Neutralizing Cross- Protective efficacy Ref Name ID mechanism neutralizing activity II CR3022 Human IgG SARS- 6YOR, Y369-N370, Trapping RBD in SARS-CoV, In the [47, 48] convalescent 6 W41 F374-K386, L390, the less stable up SARS-CoV-2 plaque-reduction patient F392, D428, T430, conformation neutralization test, F515-L517 while leading to CR3022 and destabilization of SARS-CoV-2 showed S a probit midpoint PRNT of 1:11966, corresponding to ND value of 0.114 µg/mL EY6A Human IgG Late-stage 6ZDH, Y369, F374-S375, Trapping RBD in SARS-CoV, Neutralizing  COVID-19 patient 6ZER, F377-K386, N388, the less stable up SARS-CoV-2 SARS-CoV-2 live 6ZCZ L390, P412-G413, conformation virus with ND D427-F429, L517 while leading to value of destabilization of ∼ 10.8 µg/mL VHH-72 Llama single llama immunized 6WAQ Y356-T359, Trapping RBD in SARS-CoV, Neutralizing  domain with prefusion- F361-C366, the less stable up SARS-Cov-2 pseudotyped antibody stabilized A371-T372, conformation SARS-CoV S and betacoronavirus G391-D392, R395, while leading to SARS-CoV-2 with spikes N424, I489, Y494 destabilization of IC values of S, 0.14 µg/mL Block and 0.2 mg/mL. hACE2_RBD interaction III Fab 2–4 Human IgG Infected 6XEY Y449, Y453, Locking RBD in no Neutralizing  COVID-19 L455-F456, the down SARS-CoV-2 live patients E484-F486, conformation virus with IC value Y489-F490, while occluding of 0.057 µg/mL L492-S494, G496 access to ACE2 BD23 Human IgG COVID-19- 7BYR G446, Y449, L452, Block no Neutralizing  convalescent T470, E484-F486, hACE-RBD2 SARS-CoV-2 patient Y489-F490, interaction authentic virus with L492-S494, G496, IC value of Q498, T500-N501, 8.5 µg/mL Y505 Continued 290 Antibody Therapeutics, 2020 Table 1. Continued Type Antibody Antibody type Origin PDB Epitopes Neutralizing Cross- Protective efficacy Ref Name ID mechanism neutralizing activity IV S309 Human IgG Infected SARS 6WPT, T333-L335, P337, ADCC & ADCP SARS-CoV, Neutralizing  patients 6WPS G339-V341, SARS-Cov-2 authentic N343-T345, SARS-CoV-2 (2019n- K356-C361 CoV/USA_WA1/2020) with an IC of 79 ng/ml H11-H4 Llama single Naïve llama 6ZHD K444, Y449-N450, Block no Neutralizing live  domain single-domain L452, L455-F456, hACE2-RBD SARS-CoV-2 with an antibody antibody library T470, G482-E484, interaction ND of 6 nM Y489-F490, L492-S494 H11-D4 6Z43, K444, Y449-N450, Block no Neutralizing live 6Z2M L452, L455-F456, hACE2-RBD SARS-CoV-2 with an T470, G482-E484, interaction ND of 18 nM Y489-F490, L492-S494 P2B-2F6 Human IgG COVID-19 7BWJ R346, K444, Block no Neutralizing  convalescent G446-N450, L452, hACE2-RBD SARS-CoV-2 patient V483-G485, F489, interaction pseudovirus with L491-S493 IC value of 0.05 µg/mL REGN10987 Recombinant Humanized mice 6XDG R346, N439-L441, Block no Neutralizing  full-human and COVID-19- K443-N450, Q498, hACE2-RBD SARS-CoV-2 live antibodies convalescent T500 interaction, virus with IC value patients ADCC & ADCP of 42.1 pM Antibody Therapeutics, 2020 291 Figure 2. Illustrations of four different binding modes of NAbs against SARS-CoV-2 S trimers. (A) Type-I nAbs (e.g. CB6 and REGN10933) only bind the RBD in ‘up’ states. The binding sites of CB6 and REGN10933 partially overlap with that of hACE2. (B) Type-II NAbs (e.g. EY6A, CR3022 and VHH-72) bind S trimer when at least two RBDs are in the ‘up’ state. The epitopes for this type of NAbs always distal to the binding side of hACE2. (C) Type-III NAbs (e.g. BD23 and Fab 2–4) only bind to the RBD in ‘down’ conformation. (D) Type-IV NAbs (e.g. 2F6, S309 and H11-H4) can bind both the RBDs in ‘up’ and ‘down’ conformations. 292 Antibody Therapeutics, 2020 target to the ‘down’ RBD are supposed can target to the N-glycan involving in interaction. Although there are only ‘up’RBD as the epitopes should be accessible in both states. partial overlapped binding areas between Type-IV NAbs The reason for this unusual occurrence may attribute to and the hACE2 (e.g. G446 and Y449 are the only over- the existence of N-glycans from the adjacent regions or lapping residues recognized by P2B-2F6 and hACE2), the adjacent protomers participating in the interaction with steric hindrance raised by the bound NAbs is sufficient to NAbs, and the deflection of RBD (from ‘down’ to ‘up’ block the binding of hACE2 to RBD . state) will lead to the loss of the contact between NAbs NAb S309, reported to bind to both the open and and the glycan chains. The two NAbs Fab 2–4 and Fab closed S protein, was isolated from the individual suffered 2–43 discovered by Liu et al. could neutralize live viruses from SARS-CoV in 2003 . It potently cross-neutralize with IC of 0.057 µg/mL and 0.003 µg/mL, respectively authentic SARS-CoV and SARS-CoV-2 as 17 of 22 . For Fab 2–4, The heavy chain was embedded into the residues within the epitopes and a glycan at position N343 saddle-like surface of RBD and the HCDR3 interacts with (N330 in SARS-CoV) are highly conserved. The glycan- the ridge of RBD. The N-glycan attached to N58 of heavy containing epitope is distinct from the hACE2 binding site chain form additional interactions with another N-glycan and comprises mainly α1 helices, partial β1 strand and that attached to N481 of RBD. And crucially, the Y32 on two loops spanning residues 358–361 and 333–335. These the LCDR1 also interacts with the N-glycan which located residues interact with S309 primarily through electrostatic at N343 of adjacent RBD. Fab 2–43 was observed to bind to and hydrophobic interactions. N-glycan of N343 in SARS- the same glycan-containing epitopes with a different pose CoV-2 S is a core fucose moiety, which can extend the through low-resolution map fitting, which means that the contact area for ∼300 Å by inserting into the interspace transition of RBD from ‘down’ to ‘up’ state could push between the HCDR3 and LCDR2 of S309. both the Fab 2–4 and Fab 2–43 away from the N343 glycan Another antibody REGN10987, which has developed in the adjacent ‘down’ RBD. Analogously, antibody BD23 as a therapeutic cocktail together with Type-I antibody places the heavy chain vertically in the saddle-like surface REGN10933, is also classified as Type-IV NAbs. Only the of RBD, but leave the light chain out of interaction with crystal structure of REGN10987 in complex with RBD RBD. Interestingly, an N-glycan at N165 from the NTD was determined, the structure superposition reveals that of the adjacent S protomer could facilitate the binding to REGN10987 can bind to both ‘up’ and ‘down’ RBDs BD23 and this contact will be lost when the RBD turn and the binding of REGN10987 would lead to the steric to an ‘up’ configuration . Collectively, Type-III NAbs hindrance of hACE2 binding. In the meantime, both use primarily the heavy chain to interact with RBD, with REGN10987 and REGN10933 could mediate signifi- footprints smaller and closer to the flexible ridge compared cant levels of antibody-dependent cellular phagocytosis to those of other types, and may resist dynamic instability (ADCP) and antibody-dependent cellular cytotoxicity to some extent by target a quaternary epitope that span the (ADCC), which may account for their superior antiviral RBDs and NTD. Logically, we would venture to speculate potency . The peculiar epitopes recognized by S309 that the N-glycan chains would play a key role in stabilizing and REGN10987 seems to raise another fascinating neu- the binding of Type-III NAbs to the ‘down’ RBD. In other tralizing mechanism which may cause stronger ADCP and words, the neutralization mechanism of Type-III NAbs ADCC response, S trimer cross-linking, steric hindrance or is locking RBD in the ‘down’ conformation and further aggregation of virions [40, 60]. occluding access to hACE2 . In brief, as the target of hACE2 receptor, RBD can serve The last but not least type, Type-IV NAbs, e.g. H11- as an effective immunogen to stimulate the response of D4, P2B-2F6, Ty1, S309 and REGN10987, can recognize NAbs. Most of NAbs were found targeting around the the epitopes on both the ‘up’ and ‘down’ state of RBDs top saddle-like surface of RBD and directly interfere with (Fig. 2D) [57–60]. The structural studies revealed two dif- hACE2 binding. Some other NAbs could prevent from viral ferent regions that Type-IV NAbs target to, one is located infection by trapping the RBD in ‘up’ state and destabilize on the receptor binding motif (RBM) and the other is the S protein, or lock the RBD in ‘down’ state and make located on the side of the RBD with no or little overlap with the RBM in a receptor inaccessible conformation. the RBM. In the former case, P2B-2F6 and two nanobodies H11-D4 and H11-H4 can target to the top saddle-like surface similar to NAbs from Type-III, but with a slightly NAbs TARGETING SARS-CoV-2 NTD or S2 rotated orientation and the key residues of their epitopes concentrated around the other side of RBM compared to Although RBD is the core target for NAbs against CoVs, that of Type-I, which are solvent accessible in the ‘down’ non-RBD-targeted NAbs were also reported and may state. It looks like that the binding directions of these three benefit to the strategy of NAbs cocktail therapeutics. For antibodies and those Type-I NAbs are mirror reflection SARS-CoV-2, some NAbs were reported targeting the symmetry with respect to the RBD. As a result, the protrud- NTD of spike protein, such as NAbs 4A8, COV57, 2–17, ing loop (aa. 448–452) make more essential contributions to 5–24, and 4–8 [39, 56, 62]. Specially, NAb 4A8, which was reinforce the interaction between RBD and three Type-IV isolated from Chinese convalescent patient with COVID- NAbs. Meanwhile, three antibodies take full advantage of 19, exhibits potent neutralizing activity against authentic the hydrophobic motif on RBD raised by L492, L452 and SARS-CoV-2 virus. Structural investigation revealed that F490 to form hydrophobic interactions compared to those NAb 4A8 binding could not block the interaction between of Type-III. These might explain why Type-IV antibodies hACE2 and spike protein, which is distinguished from can target to both the ‘up’ and ‘down’ RBD without extra another NAb 7D10 that targeting NTD of MERS spike Antibody Therapeutics, 2020 293 protein. 7D10 can inhibit the binding of S protein to its more promising cross-reactive epitopes when comparing cell receptor DPP4 [63, 64]. All above mentioned SARS- to the RBM. Therefore, tempting to elicit NAbs targeting CoV-2 NTD-targeting NAbs showed high potency of viral this region might provide broad and potent neutralizing neutralization, although the detailed mechanisms are still activity against CoVs. not clear. On the other hand, antibodies targeting the S2 subunit of SARS-CoV-2 spike protein have rarely reported. An mAb NAbs FOR THE TREATMENT OF COVID-19 named 1A9, generated by immunization with SARS-CoV antigen, was proved to cross-react with SARS-CoV-2 S2 Although several vaccine candidates under their clinical subunit, but have no neutralizing activity against SARS- trials and showed promising effectivities, there are no CoV-2 virus . Another NAb COV57 which derived currently available vaccines or antiviral therapeutic agents from SARS-CoV-2 immunization, was showed to recognize to the treatment of COVID-19 . A number of researches the MERS-CoV S protein . Like other CoVs includ- showed that NAbs are robust therapeutic potential against ing SARS-CoV and MERS-CoV, SARS-Co-2 S2 subunit COVID-19. Based on the existing evidence and prior mediate the fusion of viral and host cell membranes and experience in treating a novel pathogen emerges, such sequentially more conserved than S1 subunit, which raise as SARS-CoV, MERS-CoV and Influenza, convalescent the possibilities for screening broad S2 specific NAbs cross- plasma transfusion is an effective therapeutic approach react to different CoVs not only SARS-CoV and SARS- against infectious diseases [73–76]. In the early state of CoV-2. SARS-CoV-2 pandemic, convalescent plasma has been reported to be applied in treatment for patients with COVID-19. In terms of safety, transfusion of convalescent plasma has been shown with excellent safety in hospitalized BETACORONAVIRUSES CROSS-REACTIVE NAbs patients with COVID-19 [77, 78]. A recent study on the The CoVs which can infect humans (HCoVs) were classed treatment of COVID-19 patients with convalescent plasma into alpha and beta CoVs, especially the latter, such as indicated that all five patients improved their clinical status HCoV-HKU1, HCoV-OC43, MERS-CoV, SARS-CoV, accompanying viral loads decreased and became negative and SARS-CoV-2, pose serious health threats to human within 12 days after the transfusion . Similarly, another beings . For these HCoVs, the RBDs from SARS- research reported the convalescent plasma therapy was well CoV and SARS-CoV-2 have a relatively higher amino tolerated in patients with severe COVID-19, and rapidly acid identity of ∼75%, and both viruses use ACE2 as improved the clinical symptoms and characteristics of the their cell entry receptor, raising the possibility for the patients . In addition, this treatment could increase and screening of cross-neutralizing NAbs against both viruses maintain NAbs at a high level and show disappearance of [67, 68]. However, many reported SARS-CoV and SARS- virus RNA within 7 days . CoV-2 cross-reactive antibodies can only neutralize one On the other hand, NAbs serve as an alternative of this two viruses but lost the neutralization to the treatment approach against COVID-19 due to their other one, which was first demonstrated by the above- excellent neutralizing efficiency and mature industrializa- mentioned antibody CR3022 . Another antibody NAb, tion prospect. In the preclinical research, SARS-CoV-2 515–5, originating from COVID-19 patient, which can antibody CB6, the RBD-directed antibody, was reported effectively neutralize SARS-CoV-2 virus but only exhibits for the first time for the nonhuman primate therapeutics relative weaker but detectable neutralization against SARS- . CB6 administration showed strong viral inhibition CoV . Another unexpected phenomenon is that many in vivo in both prophylactic and treatment as a result SARS-CoV and SARS-CoV-2 cross-neutralizing mAbs of CB6 treatment reduced virus titers immediately after were demonstrated with no competition with hACE2, administration and the peak viral load was no more such as NAbs ADI-55689/ADI-56046, 47D11 and above- than 10 RNA copies per milliliter in pre-exposure of mentioned S309, indicating the existence of conserve SARS-CoV-2 virus. These data indicated that CB6 is epitope besides hACE2 binding sites among SARS- an outstanding candidate for translation for the clinic CoV-2 and SARS-CoV [60, 70, 71]. S309 recognizes an study. To date, many NAbs showing promising therapeutic epitope containing a conserved glycan within sarbecovirus potential have been evaluated clinically (Table 2). At least subgenus but fails to compete with hACE2 attachment. 10 monoclonal antibodies and one polyclonal antibody, Furthermore, a recent reported humanized antibody H014, were reported under different states of clinical trials. which binds a novel conformational epitope of RBD, Of these, LY-CoV555 is the first antibody to enter into efficiently neutralizes both SARS-CoV and SARS-CoV-2. phase 1 clinical trials in the world and the NAb CB6, The cryo-EM structure showed that H104 recognizes three termed as JS016 in the clinical trial, is in the steady open RBDs and critical residues involved in interaction are progress of phase 1 in China. As single antibody treatment mostly conserved and locate mainly on one side of the open may rapidly cause escape mutants on spike protein, it RBD distinct from the RBM . Similarly, other cross- is worth noting that cocktail antibodies were regarded NAbs, e.g. VHH-72, and ADI-56046, CC6.33 and COV21, as a more superior antibody therapy against SARS- most recognize the core domain (aa 318–424) of RBD CoV-2 in the preclinical study [40, 81]. For example, the other than the RBM and neutralize both viruses [45, 51, REN10987 + REGN10933 antibody cocktail that bind to 70]. The above information together suggests that the core two nonoverlapping epitopes of RBD retained the capacity domain of RBD may exhibits extensive conservation and for neutralizing all identified mutants. In general, potent 294 Antibody Therapeutics, 2020 Table 2. List of clinical-phase therapeutic NAbs candidates for COVID-19 No NCT number NAbs Clinic trail titles Phase Status Sponsor/collaborators Location 1 NCT04441918 JS016 Tolerability, Safety, 1 Recruiting Shanghai Junshi Shanghai, China Pharmacokinetic Profile Bioscience Co., Ltd and Immunogenicity of a Recombinant Humanized Anti-SARS-CoV-2 Monoclonal Antibody (JS016) for Injection in Chinese Health Subjects 2 NCT04425629 REGN10933, Safety, Tolerability, and 1,2 Recruiting Regeneron Beijing, China REGN10987 Efficacy of Anti-Spike (S) Pharmaceuticals SARS-CoV-2 Monoclonal Antibodies for the Treatment of Ambulatory Adult Patients With COVID-19 3 NCT04426695 Safety, Tolerability, and 1,2 Recruiting Beijing, China Efficacy of Anti-Spike (S) SARS-CoV-2 Monoclonal Antibodies for Hospitalized Adult Patients With COVID-19 4 NCT04519437 Study Assessing the 1 Active, not USA Safety, Tolerability, recruiting Pharmacokinetics and Immunogenicity of Repeated Subcutaneous Doses of Anti-Spike (S)SARSCoV-2 Monoclonal Antibodies (REGN10933+ REGN10987) in Adult Volunteers as Related to COVID-19 Continued Antibody Therapeutics, 2020 295 Table 2. Continued No NCT number NAbs Clinic trail titles Phase Status Sponsor/collaborators Location 5 NCT04452318 Study Assessing the 3 Recruiting USA Efficacy and Safety of Anti-Spike SARS CoV-2 Monoclonal Antibodies for Prevention of SARS CoV-2 Infection Asymptomatic in Healthy Adults Who Are Household Contacts to an Individual with a Positive SARSCoV-2 RT-PCR Assay 6 NCT04479644 BRII-198 Safety, Tolerability, and 1 Recruiting Brii Biosciences Beijing, China Pharmacokinetics Study Limited of Human Monoclonal TSB Therapeutics Antibody BRII-198 (Beijing) Co., Ltd 7 NCT04479631 BRII-196 Safety, Tolerability, and 1 Recruiting Brii Biosciences Beijing, China Pharmacokinetics Study Limited of Human Monoclonal TSB Therapeutics Antibody BRII-196 (Beijing) Co., Ltd 8 NCT04483375 SCTA01-X101 Safety, Tolerability and 1 Recruiting Sinocelltech Ltd Beijing, China Pharmacokinetics of SCTA01, an Anti-SARS-CoV-2 Monoclonal Antibody, in Healthy Chinese Subjects 9 NCT04429529 TY027 Safety of TY027, a 1 Active, Tychan Pte Ltd USA Treatment for COVID-19, not in Humans recruit- ing 10 NCT04537910 LY-CoV555, A Study of LY3819253 1 Active, Eli Lilly and USA LY-CoV016 (LY-CoV555) in Healthy not yet Company Participants recruiting 11 NCT04411628 A Study of LY3819253 1 Completed Eli Lilly and (LY-CoV555) in Company Participants Hospitalized AbCellera Biologics for COVID-19 Inc. Continued 296 Antibody Therapeutics, 2020 Table 2. Continued No NCT number NAbs Clinic trail titles Phase Status Sponsor/collaborators Location 12 NCT04497987 A Study of 3 Recruiting Eli Lilly and Company LY3819253 National Institute of Allergy (LY-CoV555) in and Infectious Diseases Preventing (NIAID) SARSCoV-2 Infection AbCellera Biologics Inc. and COVID-19in Nursing Home Residents and Staff 13 NCT04427501 A Study of 2 Recruiting Eli Lilly and Company LY3819253 AbCellera Biologics Inc. (LY-CoV555) and LY3832479 (LY-CoV016) in Participants with Mild to Moderate COVID-19 Illness (BLAZE-1) 14 NCT04634409 A Study of Immune 2 Recruiting Eli Lilly and Company System Proteins in AbCellera Biologics Inc. Participants With Shanghai Junshi Bioscience Mild to Moderate Co., Ltd COVID-19 Illness (BLAZE-4) 15 NCT04525079 CT-P59 To Evaluate the Safety, 1 Recruiting Celltrion Korea Tolerability and Pharmacokinetics of CT-P59 in Healthy Subjects 16 NCT04469179 SAB-185(polyclonal Safety, Tolerability, and 1 Recruiting SAb Biotherapeutics, Inc. USA antibody) Pharmacokinetics of Department of Health and SAB-185in Ambulatory Human Services Joint Participants With Program Executive Office COVID-19 (JPEO) 17 NCT04468958 Safety, Tolerability, and 1 Recruiting Chemical, Biological, USA Pharmacokinetics of Radiological, and Nuclear SAB-185 in Healthy Defense (CBRND) Participants Enabling Biotechnologies 18 NCT04545060 VIR-7831 VIR-7831 for the Early 2,3 Recruiting (EB) USA Treatment of COVID-19 Vir Biotechnology, Inc. in Outpatients GlaxoSmithKline Data source: https://clinicaltrials.gov/ Antibody Therapeutics, 2020 297 therapeutic antibodies or rational antibodies mixture are (REN10987 + REGN10933) directing two independent expected to be a rapid intervention against the continuing epitopes of RBD still neutralize the variants of mutated pandemic of COVID-19 in the absence of vaccines. RBD . Similarly, mixture of antibodies (C121+ C135 or C144 + C135) apparently reduce the emergence of resistance strains compared to single mAb treatment . Therefore, as more and more NAbs have been isolated, the CHALLENGES OF NAbs THERAPEUTICS more combinations of cocktail antibodies are expected to be further clinic usage. Although NAbs against SARS-CoV-2 show potent ther- apeutic potential, antibodies can also exacerbate viral infection with elusive molecular mechanisms. Antibody- dependent enhancement (ADE) is the great obstacle in the CONCLUSION development of vaccines and therapeutic antibody drugs NAbs hold excellent potential for prophylactic and ther- against some viruses. In previous clinical studies, it was apeutic applications against infectious diseases including found that the administration of vaccines of respiratory COVID-19. The process of the development of NAbs- syncytial virus (RSV) and dengue virus both cause ADE based therapeutics against SARS-CoV-2 has been expe- effects [82, 83]. For dengue virus, it is appear to worsen dited to the preclinical and clinical evaluation. NAbs disease after the second infection with viruses of other therapy should be considered as a potential candidate serotypes because of pre-existing vaccine elicited antibodies intervention for COVID-19 given that the vaccine is which have low or no cross-neutralizing activity . currently unavailable and the existence of many uncertain Additionally, non-NAb targeting the spike protein of the questions that need to be addressed in the future. In this feline infectious peritonitis virus can accelerate the viral review, we summarized the SARS-CoV-2 specific NAbs infection of macrophage in vitro . For CoVs, ADE and analysis their structures, functions and neutralization has been demonstrated occurred when antibody bound mechanisms. We provide insight into how these NAbs SARS-CoV binds to Fcγ RII of human macrophages specific recognize S protein of SARS-CoV-2 or cross-react and subsequently triggers the associated-downstream to other CoVs. We also discuss the challenges of NAbs signal . Although it is insufficient to predict the ADE therapeutics such as ADE and escape mutations. Such phenomenon of COVID-19 due to the incomplete study evidence is urgently needed to the future development evidence and fuzzy mechanisms of ADE. However, a of antibody therapeutic interventions that are required to recent study demonstrated that ADE occurs on those reduce the global burden of COVID-19. highly efficient NAbs against MERS-CoV RBD, indicating which may also occur on NAbs against SARS-CoV-2 . Therefore, it should be necessary to optimize the FUNDING animal models and further investigate the ADE risk about SARS-CoV-2. Grants from Chinese Academy of Medical Sciences SARS-CoV-2 is an RNA virus with higher mutation (CAMS) Innovation Fund for Medical Sciences (grant no. rate of its surface protein amino acids compared to those 2019RU022). DNA viruses. It is necessary to understand the relationship between NAbs and mutant viral strains. It is well demon- strated that D614G mutation of SARS-CoV-2 spike pro- CONFLICTS OF INTEREST STATEMENT tein, the major mutation detected to date, cause increased infectivity and case fatality [88, 89]. The mutation (D614G) None declared. is located between two protomers and could eliminate the contact of interprotomer . Furthermore, a recent study showed that D614G shifts S protein conformation toward REFERENCES an ACE2-binding fusion-competent state, and therefore 1. Organization, W.H, World Health Organization. WHO Coronavirus may increase the infectivity of virus . 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Antibody Therapeutics – Oxford University Press
Published: Dec 28, 2020
Keywords: SARS-CoV-2; COVID-19; spike protein; neutralizing antibodies; hACE2
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