By Oct. 29, 2020, in a matter of 10 months since the beginning of the COVID-19 outbreak, more than 43, 000, 000 confirmed cases of COVID-19 had been reported, with more than 1, 100, 000 deaths (WHO 2020). The origin and intermediate host of SARS-CoV-2, the virus responsible for COVID-19, have not been identified yet. However, it has been confirmed that this virus is mainly transmitted by respiratory droplets and direct contact (Liu Y et al. 2020). Moreover, SARS-CoV-2 viral nucleic acid was detected in the feces of patients (Wu Y et al. 2020), which has been observed in other coronavirus infection cases (Guan et al. 2015; Mackay and Arden 2015), and a rise in immunoglobulin M (IgM) antibody levels was confirmed in newborns, suggesting that SARS-CoV-2 can also be transmitted through the fecal-mouth route and vertically from mother to child (Dong et al. 2020). Humans of all ages are susceptible to SARS-CoV-2 infection, which is spreading more efficiently than influenza. Therefore, it is crucial and pressing to search for effective treatment strategies to cope with such an unprecedented health emergency.
There are currently no approved drugs or vaccines for COVID-19, highlighting the huge unmet medical need to develop treatments. Convalescent plasma therapy has been proven successful in conferring protection against SARS-CoV, MERS-CoV, avian influenza, and Ebola virus infections, considerably alleviating clinical symptoms and reducing mortality (Bloch et al. 2020). The available data also support the feasibility of this treatment for SARS-CoV-2 (Devasenapathy et al. 2020). However, a large-scale convalescent plasma transfusion program is conditioned by the limited availability of convalescent plasma and the lack of appropriate risk assessment (Shi et al. 2020). On the other hand, neutralizing monoclonal antibodies (mAbs) have attracted extensive interest as promising candidates to fight against emerging viruses. The administration of mAbs for passive immunization can provide immediate and specific protection and complement the development of preventive vaccines, thus potentially having a significant impact on the control of the COVID-19 pandemic. The successful use of mAb therapy (mAb114) during the Ebola virus outbreak highlighted the effectiveness and safety of mAbs (Corti et al. 2016). More importantly, the therapeutic potential of antibodies targeting coronaviruses was well recognized during the SARS outbreak (Wang C et al. 2020).
Therapeutic neutralizing antibodies need to go through a long process from isolation to clinical application. In vitro assays of neutralizing activity, in vivo evaluation of protective effect, and safety and clinical trials are necessary steps for antibody development. Researchers are endeavoring to develop such mAbs or their functional fragments as prophylactic or therapeutic agents to prevent or treat SARS-CoV-2 infection. Therefore, compared to the usual timelines, the development of mAbs in this pandemic setting could be reduced in 5–6 months. Moreover, the previous researches on SARS-CoV and MERS-CoV antibodies provide a basis for these studies (Wang HY et al. 2020).
47D11 was the first SARS-CoV-2-neutralizing human monoclonal antibody to be reported, which was screened from readily available hybridoma libraries of genetically modified mice that had been immunized with SARS-CoV and MERS-CoV S proteins (Wang C et al. 2020). In vitro experiments showed 47D11 could neutralize authentic SARS-CoV and SARS-CoV-2 with IC50 values of 0.19 μg/mL and 0.57 μg/mL, respectively, indicating that antibodies isolated from SARS survivors may play a huge role in our fight against SARS-CoV-2 (Table 1).
Source Antibodies Cross reactivity Blocking receptor binding Target Complex crystal (PDB ID) Binding affinity (Kd nmol/L) Neutralizing effect (IC50 μg/mL or nmol/L) Current status Refs. Convalescent SARS patients 47D11 Yes No RBD NA 9.6b 0.57† μg/mL Under research Wang C et al. (2020) S309 Yes No RBD (Site B) 6WPS 0.001b 0.079† μg/mL Under research Pinto et al. (2020) CR3022 Yes No RBD (Site C) 6W41 < 0.1b No Under research Yuan et al. (2020b) Immunized llama VHH-72-Fc Yes Yes RBD NA 38.6a 0.2* μg/mL Under research Wrapp et al. (2020a) Convalescent COVID-19 patients P2C-1F11 No Yes RBD (Site A) NA 2.12a 0.03† μg/mL Under research Ju et al. (2020) P2B-2F6 No Yes RBD (Site A) 7BWJ 5.14a 0.41† μg/mL Under research CA1 No Yes RBD (Site A) NA 4.68 ± 1.64a 4.981† μg/mL Under research Shi et al. (2020) CB6 (JS016) No Yes RBD (Site A) 7C01 2.49 ± 1.65a 0.835† μg/mL Phase-I B38 No Yes RBD (Site A) 7BZ5 70.1a 0.177† μg/mL Under research Wu Y et al. (2020) H4 No Yes RBD (Site A) NA 4.48a 0.896† μg/mL Under research 414-1 No Yes RBD (Site A) NA 0.31c 1.75† nmol/L Under research Wan et al. (2020) 505-3 No Yes RBD (Site A) NA 0.08c 3† nmol/L Under research BD-23 NA Yes RBD (Site A) 7BYR 4.3a 8.5† μg/mL Under research Cao et al. (2020) BD-368-2 NA Yes RBD (Site A) NA 0.82a 0.015† μg/mL Under research CC6.29 No Yes RBD (Site A) NA 1.20a 0.002* μg/mL Under research Rogers et al. (2020) CC6.30 No Yes RBD (Site A) NA 1.71a 0.001* μg/mL Under research CC12.1 No Yes RBD (Site A) 6XC2 5.92a 0.022* μg/mL Under research Yuan et al. (2020a) CC12.3 No Yes RBD (Site A) 6XC4 54.3a 0.026* μg/mL Under research 4A8 NA No NTD (Site D) 7C2L 1.00b 0.39† μg/mL Under research Chi et al. (2020) 2–4 NA Yes RBD (Site A) 6XEY NA 0.057† μg/mL Under research Liu LH et al. (2020) 2–15 NA Yes RBD NA NA 0.0007† μg/mL Under research 4–8 NA No NTD NA NA 0.009† μg/mL Under research 2–43 NA Yes RBD NA NA 0.003† μg/mL Under research COV2-2196 No Yes RBD NA NA 0.015† μg/mL Under research Zost et al. (2020) COV2-2130 No Yes RBD NA NA 0.107† μg/mL Under research S2M11 No Yes RBD (Site A) 7K43 66a 1.66† μg/mL Under research Poh et al. (2020) S2E12 No Yes RBD (Site A) 7K4N 1.6a 5.29† μg/mL Under research LY-CoV555 No Yes RBD NA NA NA Phase III (pause) Jones et al. (2020) REGN10933 No Yes RBD 6XDG 0.0140a 0.043† nmol/L Phase III Baum et al. (2020) VelocImmune mice REGN10987 No Yes RBD 6XDG 0.0298a 0.041† nmol/L Phase III Phage library n3088 NA No RBD (Site C) NA 12.6b 3.3* μg/mL Under research Wu YL et al. (2020) n3130 NA No RBD (Site C) NA 32.5b 3.7* μg/mL Under research n3086 NA No RBD NA 88.97b 26.6* μg/mL Under research n3113 NA No RBD NA 57.01b 18.9* μg/mL Under research H014 Yes Yes RBD (Site C) NA 0.33b 1* nmol/L Under research Lv et al. (2020) †The data were derived from authentic virus neutralization experiments
* The data were derived from the pseudovirus neutralization experiments
aThe data obtained using surface plasmon resonance (SPR)
bThe data obtained using biolayer interferometry (BLI)
cThe data obtained using enzyme-linked immunosorbent assay (ELISA)
*The data were derived from the pseudovirus neutralization experiment (other data derived from authentic virus neutralization experiments)
Table 1. Characteristics of representative antibodies to SARS-CoV-2.
Many neutralizing antibodies derived from B cells of patients infected with SARS-CoV have been reported until now, such as S309 (Pinto et al. 2020), CR3022, CR3014 (Yuan et al. 2020b), 80R (Hwang et al. 2006), and m396 (Prabakaran et al. 2006), some of which showed potent cross-reactivity to SARS-CoV-2. S309 can neutralize both SARS-CoV and SARS-CoV-2, while CR3022, CR3014, 80R, and m396 can only neutralize SARS-CoV. IC50 value of S309 for SARS-CoV-2 neutralization was 0.079 μg/mL. Interestingly, CR3022 also showed cross-reactivity with SARS-CoV-2 RBD (Table 1).
In addition, Wrapp et al. obtained a single-domain antibody (VHH), VHH-72, derived from a llama immunized with SARS-CoV and MERS-CoV S proteins, that can bind tightly to the RBD region of SARS-CoV S protein and neutralize SARS pseudoviruses. By further engineering this VHH into a bivalent Fc-fusion protein, they showed that this novel bivalent molecule could also neutralize SARS-CoV-2 pseudoviruses with an IC50 of approximately 0.2 μg/mL (Wrapp et al. 2020a) (Table 1).
As with other human pathogens, mAb can be easily isolated from the B cells of patients infected with SARS-CoV-2 by the antigenic protein bait. Thus, the protein bait needs to be functionally relevant and pure enough to isolate antibodies that are relevant to the epitopes on the native spike. The RBD domain and S1 subunit of SARS-CoV-2 are often selected as protein bait for fishing out the antibodies. Severe cases seem to have a much higher neutralizing antibody response and hence more likelihood of obtaining potent neutralizing antibodies (Zhao et al. 2020). However, it is unclear about the relationship between the severity of diseases and antibody response. It will take a while to figure out the cause and consequence of the two.
Ju et al. first analyzed the immune responses of eight SARS-CoV-2-infected patients after exposure to virus stimulation, including the level of viral-specific antibodies in plasma and the proportion of specific B cells in memory cells, finding that the antibody response levels among these infected patients varied greatly (Ju et al. 2020). Further, this group isolated 206 monoclonal antibodies capable of recognizing SARS-CoV-2 RBD and obtained their paired heavy- and light-chain gene sequences. Using a pseudovirus evaluation system, the antibody inhibition rate of 12 strains exceeded 50% and that of the other seven strains exceeded 80%. Among them, P2C-1F11 and P2B-2F6 were elite antibodies with IC50 of 0.03 μg/mL and 0.05 μg/mL, respectively (Table 1).
Moreover, Shi et al. used SARS-CoV-2 S protein recombinant RBD as bait to screen specific memory B cells in peripheral blood mononuclear cells (PBMCs) from peripheral blood of convalescent patients with COVID-19 (Shi et al. 2020). The variable region sequences of these B cell receptors were cloned into a vector containing a constant region to produce a series of IgG antibodies. Among them, CB6 was selected for testing in animal models and the results showed that this antibody not only reduced the virus titer but also inhibited the pathological lung damage in the monkeys challenged with SARS-CoV-2.
Meanwhile, a team led by George Fu Gao isolated four human mAbs (B38, H4, B5 and H2) from a rehabilitated patient, all of which have shown in vitro neutralization of SARS-CoV-2 and two of which (B38 and H4) block the binding of RBD to the cellular receptor ACE2 (Wu et al. 2020b) (Table 1).
On the other hand, Wan et al. enriched more than 1000 antigen-specific B cells in plasma from 11 COVID-19 rehabilitation patients by using the recombinant S1 or RBD antigen of SARS-CoV-2 and cloned 729 pairs of naturally paired antibody genes of single B cells (Wan et al. 2020). The team mainly reported 11 different neutralizing antibody sequences, eight of which showed an IC50 within 10 nmol/L and the best one, 414-1, with an IC50 of 1.75 nmol/L. Besides, 553-15 could be combined with other neutralizing antibodies and increase their neutralizing ability (Table 1).
Meanwhile, Cao et al. used high-throughput cell sequencing to identify 14 highly active neutralizing antibodies from a screening of 8558 viral protein-binding antibody sequences, the most effective of which was a neutralizing antibody called BD-368-2 (Cao et al. 2020). Unlike traditional methods, large-scale data obtained from high-throughput single cell can help researchers observe the clonal enrichment of B cells before antibody expression in vitro and calculate the degree of enrichment based on the number of cloned cells observed. The researchers suggest that enriched B cell clones are more likely to produce novel binding SARS-CoV-2 neutralizing antibodies with high affinity. In addition, by using bioinformatics methods to predict the structure of CDR3H, researchers found mAbs with highly similar CDR3H structures to the SARS-CoV neutralizing antibody m396 showed a high neutralization potency for SARS-CoV-2. The largely conserved combination of the VDJ gene segment and the likely overlapping epitopes of those mAbs suggest the existence of stereotyped B cell receptors against SARS-CoV-2 (Cao et al. 2020). A new study supports this idea: Yuan et al. compiled a list of 294 SARS-CoV-2 RBD-targeting antibodies where information on IGHV gene usage is available and found that IGHV3-53 is the most frequently used IGHV gene among these antibodies (Yuan et al. 2020a) (Table 1).
Furthermore, Chi et al. isolated and identified mAbs from 10 recovering COVID-19 patients. Three of these mAbs showed neutralizing activity against SARS-CoV-2. 4A8 showed high potency against both SARS-CoV-2 and its pseudovirus and it works by binding to a unique region in the NTD rather than RBD of the S protein (Chi et al. 2020) (Table 1).
To study the antibody response against SARS-CoV-2 and discover mAbs, the Scripps research team adapted their pipeline to rapidly isolate and characterize mAbs from convalescent donors (Rogers et al. 2020). A group of SARS-CoV-2 donors who had previously tested positive on the swab test was recruited for PBMC and plasma collection. Assessed by the neutralizing activity of convalescent serum against SARS-CoV and SARS-CoV-2 and selected eight donors for mAb discovery. They classified single antigen-specific memory B cells and recovered and cloned the corresponding variable genes using a high-throughput production system that could achieve antibody expression and identification within 2 weeks. More than 1000 mAbs were isolated from three convalescent donors by memory B cell selection using SARS-CoV-2 S or RBD recombinant proteins. Notably, the most potent neutralizing antibodies were those directed to the core region of RBD, known as the receptor-binding motif (RBM), including two antibodies, CC6.29 and CC6.30, that neutralize SARS-CoV-2 pseudovirus with an IC50 of 2 ng/mL and 1 ng/mL, respectively (Table 1).
Liu LH et al. (2020) isolated 61 SARS-CoV-2 neutralizing antibodies from 5 severe COVID-19 patients. Among them, nine antibodies showed very high neutralization potency, with IC50 values ranging from 0.7 to 9 ng/mL. The nine antibodies are diverse and can be divided into three categories, four against RBD (2–15, 2–7, 1–57, and 1–20), three against NTD (2–17, 5–24, and 4–8), and two against the undetermined region of S trimer (2–43 and 2–51). Notably, 2–15, binding to the top region of RBD, exhibits the most competitive against ACE2 and the most neutralizing activity to SARS-CoV-2 (Table 1).
In addition, Wu YL et al. (2020) developed a new technique to screen and reconstruct the full human heavy-chain variable region antibody skeleton. Using this technique, a whole human nanometer antibody library based on natural germline genes was designed. The RBD domain and S1 protein subunit of SARS-CoV-2 were selected as antigens, and nano-antibodies targeting multiple epitopes were selected from the antibody library. Among them, antibodies n3130, n3088, n3086, and n3113 showed moderate neutralization activities, inhibiting SARS-CoV-2 pseudovirus infection in a dose-dependent manner with IC50 values of 26.6, 18.9, 3.3, and 3.7 μg/mL, respectively. The combination of n3088 or n3130 with n3113 showed synergistic neutralization of SARS-CoV-2, with IC50 values of 0.51 and 0.70 μg/mL, respectively. Similarly, Lv et al. recently established a coronavirus antibody library by using phage display technology and identified an antibody, H014, with broad-spectrum neutralization of the coronavirus family by high-throughput screening (Lv et al. 2020). Pseudovirus neutralization assays revealed that H014 has a potent neutralizing activity, with an IC50 of 3 nmol/L and 1 nmol/L against SARS-CoV-2 and SARS-CoV pseudoviruses, respectively (Table 1).
With the advancement of research, many antibody drugs have entered clinical trials. It is worth mentioning that on June 5, 2020, the China Food and Drug Administration officially approved the first clinical trial of a novel therapeutic antibody, mAb CB6 derived from a convalescent patient, which is now at the edge of phase I. Additionally, on June 11, 2020, REGN announced the clinical trial of its antibody cocktail therapy for the treatment and prevention of COVID-19. It contains two antibodies, REGN10933 and REGN10987, which were obtained by regeneration from the VelocImmune transgenic mouse platform and COVID-19 rehabilitation patient blood based on the screening of the efficacy and binding ability of the S protein (Baum et al. 2020). Moreover, more than 10 antibody drugs such as LY-CoV555 (phase III), AZD7442 (phase I), CT-P59 (phase I) and TY027 (phase I) are already in clinical trials (https://clinicaltrials.gov/), giving us the hope of dealing with the COVID-19.