
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39258934
2404166
10.1080/22221751.2024.2404166
Version of Record
Emerging and Re-Emerging Coronaviruses
Research Article
Bispecific antibodies provide broad neutralization of emerging beta-coronaviruses by targeting ACE2 and viral spikes
Emerging Microbes & Infections
M. Li et al.
Li Minghui a#
Zhao Chaoyue a#
Shi Jialu b#
Wang Xun a#
Liu Yuanchen b
Zhao Xiaoyu a
Cai Guonan a
Chu Hin b
https://orcid.org/0000-0003-2454-7652
Wang Pengfei a
a Shanghai Pudong Hospital, Fudan University Pudong Medical Center, State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, People’s Republic of China
b State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, People’s Republic of China
CONTACT Hin Chu hinchu@hku.hk
Pengfei Wang pengfei_wang@fudan.edu.cn
# These authors contributed equally

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2404166.

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© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 have recurrently emerged as significant pathogens, causing severe respiratory illnesses and presenting challenges to monoclonal antibody therapeutics due to their rapid evolution, particularly the diverse variants of SARS-CoV-2. In this study, we utilized “Knob-into-Hole” and “IgG-scFv” technologies to engineer bispecific antibodies (bsAbs) that target both the viral receptor and spike protein, enhancing their neutralization breadth and potency. Our bsAbs, combining anti-SARS-CoV-2 or anti-MERS-CoV antibodies with an anti-ACE2 antibody, demonstrated effective neutralization across a range of SARS-CoV-2 variants, SARS-CoV and MERS-CoV in both pseudovirus and authentic virus assays. Notably, the “IgG-scFv” bsAbs format exhibited superior binding and neutralization capabilities compared to the “Knob-into-Hole” configurations. The most effective of these, “IgG-scFv” H11B11_m336, displayed exceptional neutralization potency against a panel of 24 pseudotyped Beta-Coronaviruses, with IC50 values ranging from 0.001–0.183 μg/mL. Overall, our findings underscore the potential of bsAbs as an effective strategy to meet the immediate challenges posed by existing and emerging pathogens, thereby enhancing global pandemic preparedness.

KEYWORDS

SARS-CoV-2
SARS-CoV
MERS-CoV
beta-coronavirus
bispecific antibody
broadly neutralizing antibody
Program of Science and Technology Cooperation with Hong Kong, Macao and Taiwan 23410760500 Xiaomi Young Talents Program National Natural Science Foundation of China Excellent Young Scientists Fund (Hong Kong and Macau) 32122001 AI for Science project of Fudan University XM06231724 National Natural Science Foundation of China 10.13039/501100001809 32270142 Shanghai Rising-Star Program 10.13039/501100013105 22QA1408800 This study was supported by funding from the National Natural Science Foundation of China (32270142 to P.W.), Shanghai Rising-Star Program (22QA1408800 to P.W.), and the Program of Science and Technology Cooperation with Hong Kong, Macao and Taiwan (23410760500 to P.W.). Pengfei Wang acknowledges support from AI for Science project of Fudan University (XM06231724) and Xiaomi Young Talents Program. Hin Chu acknowledges funding from National Natural Science Foundation of China Excellent Young Scientists Fund (Hong Kong and Macau) (32122001).
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pmcIntroduction

Seven human coronaviruses (HCoVs) have been reported up to date, including four common cold causing viruses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1), which have been circulating among humans for a long time. The other three includes viruses responsible for the severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the COVID-19 pandemic, all of which can lead to severe respiratory illness and have caused significant global health emergencies. Both the severe acute respiratory syndrome coronavirus (SARS-CoV) and the more recent SARS-CoV-2 belong to the beta-coronavirus family and utilize human angiotensin-converting enzyme 2 (ACE2) as their receptor [1]. The SARS outbreak resulted in over 8,000 infections and over 700 deaths globally [2]. Because of the higher transmissible and immune escape properties, SARS-CoV-2 has caused over 700 million infection cases with over 7 million deaths [3]. As of today, the original SARS-CoV-2 virus has evolved into multiple distinct variants. The Omicron variant contains over 30 mutations in its spike (S) protein and has given rise to numerous subvariants including BA.2, BA.4, BA.5, BF.7, BQ.1, and the recent XBB and BA.2.86 sub-lineages, which led to substantial changes in infectivity, antigenic escape, antibodies resistance and increased possibility of repeat infections [4–7]. MERS-CoV, another beta-coronavirus using receptors other than ACE2, has previously posed a great threat to human health [1, 8, 9]. In addition, the increasing risk of zoonotic spillovers underscores the need for developing interventions to effectively address coronaviruses with pandemic potential [10, 11]. Therefore, creating potent, broad-spectrum neutralizing antibodies targeting beta-coronaviruses is crucial.

Antibodies play a crucial role in prophylaxis and therapy against viral infections, particularly for individuals with compromised immune systems. Monoclonal antibodies (mAbs) have been isolated for SARS-CoV [12–14] and MERS-CoV [15], as well as SARS-CoV-2 and its variants [16–27]. However, the Spike (S) glycoprotein of SARS-CoV-2 has undergone numerous genomic mutations, especially in the receptor-binding domain (RBD) and the N-terminal domain (NTD), reducing the effectiveness of most mAbs. For example, Brii-196 (amubarvimab), approved in China alongside romlusevimab, maintained low activity against subvariants like BA.5 and BF.7 but was ineffective against BA.2.75.2, XBB.1, XBB.1.5, BQ.1, and BQ.1.1 [17]. LY-CoV1404 (bebtelovimab), the only mAb with clinical authorization/approval, retained neutralization activity against BA.1, BA.2, and BA.3 in our previous studies [16], but was ineffective against XBB and BQ variants [16, 28]. S309 (sotrovimab), an mAb derived from an individual infected with SARS-CoV [18], exhibited broad neutralizing activity against SARS-CoV and SARS-CoV-2 but showed reduced activity against rapidly emerging variants. Research also indicates that antibodies targeting the more conserved S2 region in coronaviruses, such as S2P6 [29] and CC40.8 [30], have greater potential for broad-spectrum neutralization. These antibodies, along with others targeting the fusion peptide (FP) region [31–33], offer promising avenues for broad neutralization of beta-coronaviruses, including MERS-CoV. However, the current neutralizing potency of these S2 antibodies is low, necessitating the development of more effective broadly neutralizing antibodies to combat multiple coronaviruses.

Bispecific antibodies (bsAbs), ingeniously crafted with one arm aimed at the virus and another at the cell receptor, introduce a cutting-edge approach to combating viral infections. By engaging the viral antigen, one segment of these dual-function antibodies neutralizes the virus, thwarting its attachment and entry into host cells. Simultaneously, the second arm impedes the virus-receptor interaction by targeting a specific cell receptor, thereby bolstering the host's defense against the infection. Such bispecific antibodies have been explored in targeting viruses, notably HIV-1 [34]. In this study, we've curated a comprehensive bsAb library utilizing the “Knob-into-Hole” CrossMab or IgG-scFv methods, pairing parental anti-SARS-CoV-2 antibodies (2-36, Brii-196, DH1047, S309, S3H3, and S2P6) or anti-MERS-CoV antibodies (m336) with the anti-ACE2 antibody, H11B11 [35, 36]. This endeavour yielded bsAbs showcasing remarkable neutralization potency and breadth against a spectrum of sarbecoviruses and even broader beta-coronaviruses, including MERS-CoV, underscoring their potential as promising therapeutic candidates.

Results

“Knob-into-Hole” bsAbs neutralizing SARS-CoV-2 variants and SARS-CoV

Building on our experience with constructing anti-HIV-1 bsAbs targeting the viral envelope and the CD4 receptor through the “Knob-into-Hole” CrossMAb technology – achieving potent and broad neutralization against a wide array of HIV-1 strains [34, 37] – we embarked on developing new bsAb candidates aimed at neutralizing SARS-CoV-2 variants and SARS-CoV. These candidates feature one arm targeting the ACE2 receptor, utilized by both SARS-CoV and SARS-CoV-2 for entry, and another arm comprising anti-SARS-CoV-2 antibodies targeting various segments of the SARS-CoV-2 S trimer, including four that target the RBD (2-36, Brii-196, DH1047, S309) and two that target regions outside the RBD (S3H3 and S2P6) (Figure 1(A)). By integrating the ACE2-targeting antibody, H11B11, into either the Knob arm or the Hole-Cross arm, we curated a library of 12 bsAbs. The resulting bsAbs, named H11B11_2-36, H11B11_Brii-196, H11B11_DH1047, H11B11_S309, H11B11_S3H3, and H11B11_S2P6, denote the positioning of H11B11 in the Knob arm. Additionally, we alternated H11B11 to the Hole-Cross arm, crafting another six bsAbs correspondingly. Size Exclusion Chromatography (SEC) was employed to assess the purity of these bsAbs, revealing only single homogeneous peaks, which indicates successful expression and purification of our bsAbs (Figure S1(A)). Figure 1. Neutralization of SARS-CoV-2 variants and SARS-CoV by “Knob-into-Hole” bsAbs. (A) Schematic diagrams illustrating the structure of “Knob-into-Hole” bsAbs, with one arm targeting the receptor and the other targeting the SARS-CoV-2 S trimer. The anti-receptor and anti-viral arms are depicted in blue and orange, respectively. (B) Neutralization IC50 titres of the bsAbs against SARS-CoV-2 variants (WT, BA.2, BA.5, XBB.1.5) and SARS-CoV. (C) Comparative analysis of IC50 values for different bsAb designs. H11B11_* and *_H11B11 denote the positioning of the H11B11 arm in the “Knob” and “Hole-Cross’ configurations, respectively. (D) Binding activities of H11B11_Brii-196 and Brii-196_H11B11 to the SARS-CoV-2 S trimer and sACE2 protein. (E) Neutralization of representative SARS-CoV-2 WT and BA.5 pseudoviruses by H11B11_Brii-196, alongside parental and single-arm antibodies. (F) Neutralization activity of HP6H8_Brii-196 and its parental antibodies against various SARS-CoV-2 variants and SARS-CoV.

To explore their capabilities, we first conducted a neutralization assay against five representative pseudoviruses, including SARS-CoV-2 wild-type (WT/D614G), BA.2, BA.5, XBB.1.5, and SARS-CoV. The neutralization curves are presented in Figure S1(B), and the half-maximal inhibitory concentration (IC50) values are displayed in Figure 1(B). The results demonstrate that all the bsAbs we engineered effectively neutralize all five pseudoviruses with potencies ranging from 0.054–3.625 μg/mL. This indicates that our strategy of combining two types of antibodies – one targeting the receptor and the other targeting the viral spike trimer – into a single bsAb is effective. When comparing the two sets of bsAbs – those with H11B11 located in the Knob arm (H11B11_*) and those in the Hole-cross arm (*_H11B11) – the bsAbs with H11B11 in the Knob arm exhibited superior neutralization activity against all five viruses, as evidenced by generally lower IC50 values (Figure 1(C)).

We next sought to determine how each arm of the bsAbs contributes to their overall activity. We conducted ELISA assays to assess the binding of the bsAbs to soluble ACE2 (sACE2) protein and the SARS-CoV-2 S trimer (Figure 1(D) and Figure S2). The results confirmed that both arms of the bsAbs are functional, as they successfully bound to sACE2 (directed by H11B11) and the SARS-CoV-2 S trimer (directed by SARS-CoV-2 antibodies). This validation confirms the successful expression of our bsAbs from a binding perspective. Notably, H11B11 in the Knob arm exhibited stronger binding to the sACE2 protein compared to when it was in the Hole-cross arm, which likely explains the enhanced neutralization potency of the H11B11_* bsAbs. Among these bsAbs, we selected H11B11_Brii-196, which showed the highest neutralization activity, for further comparison against its parental antibodies, the mixture of the parental antibodies, as well as two single-arm antibodies: one pairing H11B11 with an irrelevant anti-HIV-1 antibody, PGDM1400 [38], and the other pairing Brii-196 with another irrelevant anti-HIV-1 antibody, 3BNC117 [39] (Figure 1(E)). For the SARS-CoV-2 WT virus, while both the parental and single-arm antibodies (H11B11_PGDM1400 and Brii-196_3BNC117) neutralized the virus, the single-arm antibodies exhibited significantly lower activities, indicating the importance of avidity in neutralization. For the BA.5 variant, which is less sensitive to Brii-196, the single-arm Brii-196 completely lost its activity. However, the bsAb H11B11_Brii-196 maintained substantially better activity against the BA.5 variant compared to the single-arm H11B11 (H11B11_PGDM1400), suggesting that the Brii-196 arm significantly contributes to the bsAb's neutralization activity. Overall, these findings confirm that both arms of the “Knob-into-Hole” bsAbs contribute effectively, highlighting the synergistic effect achieved by combining two arms in a bsAb for optimal performance.

CD147 has been identified as a receptor for SARS-CoV-2, and HP6H8 is an anti-CD147 antibody known to neutralize the virus [40, 41]. We, therefore, engineered a bsAb by combining HP6H8 with Brii-196 using the “Knob-into-Hole” format and assessed its neutralizing capability (Figures 1(A) and (F)). While HP6H8 alone showed no neutralization activity in our system, its combination with Brii-196 in a bsAb format demonstrated some degree of activity, even against the SARS-CoV-2 XBB.1.5 variant and SARS-CoV, which neither parental antibody could neutralize. This, again, underscores the synergistic effect of the two arms. However, due to the relatively low activity of this bsAb, our further investigations focused exclusively on bsAbs containing H11B11.

“Knob-into-Hole” bsAbs neutralizing SARS-CoV-2 variants, SARS-CoV and MERS-CoV

Inspired by the positive results and the fact that the single-arm H11B11 exhibits robust neutralizing activity against both SARS-CoV-2 variants and SARS-CoV, we ventured to broaden the spectrum of activity to include MERS-CoV. To this end, we engineered “Knob-into-Hole” CrossMab bsAbs by pairing H11B11 with an anti-MERS-CoV antibody, m336 [15]. We created two configurations: H11B11_m336, with H11B11 in the Knob arm, and m336_H11B11, with H11B11 in the Hole-cross arm, both of which were expressed and purified to high quality (Figure 2A). ELISA assays confirmed that each bsAb could bind effectively to sACE2 and MERS-CoV S Trimer, demonstrating the functionality of both arms. For both H11B11 and m336, they displayed higher binding affinities to their target proteins when positioned in the Knob arm (Figure 2(B)), a pattern that was mirrored in their neutralizing activities (Figure 2(C)). Both bsAbs were capable of neutralizing the tested SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. Notably, H11B11_m336 exhibited stronger neutralization against SARS-CoV-2 variants (WT, BA.2, BA.5, XBB.1.5) and SARS-CoV, comparable to the mixture of the two parental antibodies, suggesting superior activity of H11B11 in the Knob arm for ACE2-utilizing viruses. Conversely, m336_H11B11 demonstrated enhanced neutralization of MERS-CoV, with IC50 values of 0.051 μg/mL – approximately four times more potent than H11B11_m336 and comparable to the mixture of the two parental antibodies, indicating that m336 positioned in the Knob arm optimizes activity against MERS-CoV (Figure 2(C)). Figure 2. Neutralization of SARS-CoV-2 variants, SARS-CoV, and MERS-CoV by “Knob-into-Hole” bsAbs. (A) SEC analysis of the “Knob-into-Hole” bsAbs – H11B11_M336 and M336_H11B11, demonstrating the purity and monodispersity of these antibody constructs. (B) Binding activities of H11B11_M336 and M336_H11B11 to the MERS-CoV S trimer and sACE2 protein. (C) Comparative neutralization by H11B11_M336, M336_H11B11 and the mixture of H11B11 and M336, against SARS-CoV-2 variants (WT, BA.2, BA.5, XBB.1.5), SARS-CoV, and MERS-CoV. (D) Neutralization of representative SARS-CoV-2 WT, BA.5, and MERS-CoV pseudoviruses by H11B11_M336, alongside parental and single-arm antibodies.

We subsequently assessed the neutralization performance of H11B11_m336 compared to its single-arm and parental counterparts to evaluate the contribution of each antibody arm. The neutralization capacity of the single-arm H11B11 (H11B11_PGDM1400) was comparable to the bsAb H11B11_m336 against SARS-CoV-2 WT and BA.5, suggesting that the efficacy of the bsAb against ACE2-utilizing viruses primarily resulted from the H11B11 component (Figure 2(D)). This is expected, given that m336 does not neutralize SARS-CoV-2. Parental H11B11 displayed enhanced activity compared to H11B11_m336 against SARS-CoV-2 viruses, highlighting that avidity enhances H11B11’s effectiveness. Similarly, parental m336 demonstrated significantly better performance against MERS-CoV than H11B11_m336. Intriguingly, the single-arm m336, when paired with an irrelevant 3BNC117, performed better than when paired with H11B11, for reasons yet to be elucidated (Figure 2(D)). Taken together, our approach successfully expanded the neutralization breadth to encompass SARS-CoV-2 variants, SARS-CoV, and MERS-CoV by integrating anti-ACE2 and anti-MERS antibodies into a single bsAb. However, the placement of the antibodies in the Knob or Hole-cross arm, along with the impact of avidity, significantly influenced their efficacy, prompting further optimization and refinement of the bsAb design.

“IgG-scFv” bsAbs exhibited better neutralization activity than “Knob-into-Hole” bsAbs

We then developed a new bsAb format, the “IgG-scFv” format, by fusing the single-chain variable fragments (scFvs) of parental antibodies to the C-terminus of a standard IgG antibody. This configuration allows each antibody to retain two binding arms, potentially overcoming the avidity limitations encountered with the “Knob-into-Hole” format. Drawing from our previous work, we paired H11B11 with Brii-196 or m336 to create four “IgG-scFv” bsAbs: H11B11_Brii-196 and H11B11_m336, where scFvs of Brii-196 or m336 were attached to the C-terminus of IgG H11B11, as well as Brii-196_H11B11 and m336_H11B11, with scFv H11B11 positioned at the C-terminus (Figure 3(A)). All four “IgG-scFv” bsAbs were expressed with satisfactory purity (Figure S3(A)). Non-reduced and reduced SDS-PAGE analysis confirmed the correct molecular size for these “IgG-scFv” bsAbs, with heavy chains approximately 70 kDa, larger than the ∼50 kDa heavy chain size observed in the “Knob-into-Hole” bsAbs (Figure S3(B)). Figure 3. Neutralization of SARS-CoV-2 variants, SARS-CoV, and MERS-CoV by “IgG-scFv” bsAbs. (A) Schematic diagrams of “IgG-scFv” bsAbs structures, showcasing anti-viral antibodies (Brii-196 or m336) linked in the scFv format to the C-terminus of the anti-ACE2 H11B11 IgG, or vice versa. (B) Binding activities of the four “IgG-scFv” bsAbs to the SARS-CoV-2/MERS-CoV S trimer and sACE2 protein. (C) Neutralization of the four “IgG-scFv” bsAbs against the representative SARS-CoV-2 (WT, BA.2, BA.5, XBB.1.5), SARS-CoV and MERS-CoV pseudoviruses. (D) Comprehensive neutralization profile of the four “IgG-scFv” bsAbs and three parental antibodies against an expanded panel of 24 pseudoviruses, including additional SARS-CoV-2 variants and animal coronaviruses. (E) Comparative analysis of the neutralization efficacy of H11B11_m336 in the “IgG-scFv” format versus the “Knob-into-Hole” format.

The “IgG-scFv” bsAbs demonstrated strong binding to both the ACE2 protein and their respective viral targets, including either the SARS-CoV-2 or MERS-CoV S trimers, as determined by ELISA using soluble proteins (Figure 3(B)) and by FACS using cell surface-expressed proteins (Figure S4). Compared to the above binding results, these bsAbs exhibited higher binding affinities compared to their “Knob-into-Hole” counterparts, potentially due to their multivalent binding capabilities with sACE2 or the S trimers. To evaluate their neutralizing efficacy, we initially tested the inhibition of coronavirus infection using six representative pseudoviruses: SARS-CoV-2 (WT, BA.2, BA.5, XBB.1.5), SARS-CoV, and MERS-CoV (Figure 3(C)). The remarkable neutralization potency and breadth of these bsAbs encouraged us to extend our assay to include 24 pseudoviruses, encompassing more SARS-CoV-2 variants including previously identified VOCs (B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1), emerging variants such as EG.5.1, and animal sarbecoviruses like GD-Pangolin and WIV1 (Figures S3(C) and (D)). The “IgG-scFv” bsAbs, H11B11_Brii-196 and Brii-196_H11B11, neutralized all tested SARS-CoV-2 sub-lineages, SARS-CoV, and animal sarbecoviruses with IC50 values ranging from 0.001–0.768 μg/mL, but, as expected, not MERS-CoV. Both “IgG-scFv” H11B11_m336 and m336_H11B11 also showed effective neutralization against all viruses, with H11B11_m336 (IC50 values ranging from 0.001–0.183 μg/mL) exhibiting superior potency compared to m336_H11B11. Importantly, both “IgG-scFv” H11B11_m336 and m336_H11B11 displayed extremely high potency against MERS-CoV, achieving IC50 values of approximately 0.001 μg/mL. While the parental H11B11 neutralized only ACE2-using SARS-CoV or SARS-CoV-2-like sarbecoviruses, and the parental m336 neutralized only MERS-CoV, combining them into bsAbs broadened the coverage to effectively neutralize all these viruses. When comparing the neutralization efficacy of H11B11_m336 in the “IgG-scFv” format to its performance in the “Knob-into-Hole” format against the six viruses tested in both formats (Figure 3(E)), it was evident that the “IgG-scFv” configuration of H11B11_m336 significantly enhanced its overall activity.

Given the outstanding neutralization activity of the “IgG-scFv” bsAbs against pseudoviruses, we extended our testing to authentic viruses, including various SARS-CoV-2 variants (WT, BA.1, XBB.1, EG.5.1, BA.2.86), SARS-CoV (GZ50), and MERS-CoV (EMC/2012). Consistent with the pseudovirus results, H11B11_Brii-196 and Brii-196_H11B11 effectively neutralized all tested SARS-CoV-2 variants and SARS-CoV but did not neutralize MERS-CoV. While the parental H11B11 and m336 could not neutralize all the viruses, both H11B11_m336 and m336_H11B11 efficiently neutralized all viruses, including MERS-CoV (Figure 4(A)). H11B11_m336 demonstrated superior neutralization potency against SARS-CoV-2 variants and SARS-CoV, while m336_H11B11 showed enhanced potency against MERS-CoV (Figure 4(B)). Taken together, these results confirm that the “IgG-scFv” bsAbs, by combining receptor-targeting and virus-targeting antibodies, achieve broad-spectrum virus neutralization through multi-target inhibition and increased avidity. Figure 4. Authentic virus neutralization by “IgG-scFv” bsAbs of SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. (A) Neutralization curves depicting the effectiveness of the four “IgG-scFv” bsAbs and three parental antibodies against authentic viruses of SARS-CoV-2 (WT, BA.1, XBB.1, EG.5.1, BA.2.86), SARS-CoV, and MERS-CoV. (B) Comparative analysis of the neutralization efficacy of “IgG-scFv” bsAbs, contrasting H11B11_Brii-196 with Brii-196_H11B11 and H11B11_m336 with m336_H11B11.

Discussion

Coronaviruses, particularly pathogenic strains such as SARS-CoV, MERS-CoV, and SARS-CoV-2, have triggered significant global health crises. The rapid emergence of new variants, such as the Omicron sub-lineages evolving from SARS-CoV-2, underscores the urgent need for adaptable and innovative therapeutic strategies. Monoclonal antibodies have played a critical role in prophylaxis and therapy against these viral infections, offering vital protection, especially for immunocompromised individuals. However, the continuous genetic drift observed in the viral proteins of these viruses presents challenges to the effectiveness of existing antibodies.

Our exploration into bsAbs represents a significant stride towards addressing this challenge. BsAbs, capable of simultaneously targeting two distinct antigens or antigenic sites [42], benefit from the synergistic effects of dual binder arms [43]. Existing bsAbs that combine two anti-SARS-CoV-2 antibodies have demonstrated efficacy against Omicron variants [43–46]. In this study, we have engineered bsAbs that concurrently target both the virus and the cell receptor, promising enhanced dual-action neutralization efficacy. Our previous work has shown that ACE2-targeting antibodies like 3E8 and H11B11 retain potent neutralization against all Omicron subvariants [47], affirming the potential of the ACE2 receptor as one arm of the bsAb. Despite trials with 3E8, its low productivity and suboptimal neutralization prompted us to select the more effective H11B11. Although CD147 has been implicated in SARS-CoV and SARS-CoV-2 infections as an alternative receptor [40, 41], the minimal neutralization achieved with the anti-CD147 antibody HP6H8 discouraged further exploration. By combining the receptor-targeting specificity of antibodies like H11B11 with the viral neutralization capabilities of antibodies such as Brii-196 and m336, we aim to harness synergistic effects that could offer a broader spectrum of protection against a range of coronaviruses, including challenging variants that might evade single-target antibodies.

Initially, we developed a “Knob-into-Hole” bsAb library and observed that while both arms functioned effectively, the placement of the antibodies in the Knob or Hole-cross arm significantly impacted performance, and for some antibodies, avidity was crucial (Figures 1 and 2). This led us to refine our approach, advancing to the “IgG-scFv” format, which facilitated broad-spectrum virus neutralization through multi-target inhibition and enhanced avidity (Figures 3 and 4). This innovation in antibody design aims to overcome the challenges posed by viral mutations and immune evasion strategies.

The promising results from this study highlight the potential of bsAbs to neutralize not just SARS-CoV-2 and its variants but to extend to other beta-coronaviruses, including MERS-CoV. The comprehensive neutralization profiles of these bsAbs, particularly against virulent strains and their maintained efficacy across various mutations, position them as formidable candidates for future therapeutic development. This strategy not only addresses the immediate threats posed by current pandemic strains but also lays the groundwork for responding to potential future zoonotic spillovers.

Materials and methods

Cell lines

Expi293F cells (Thermo Fisher Cat# A14527) were cultured in the serum free SMM 293-TI medium (Sino Biological Inc.) at 37°C with 8% CO2 on an orbital shaker platform. HEK293 T cells (cat# CRL-3216), Vero E6 cells (cat# CRL-1586) were from ATCC and cultured in 10% Fetal Bovine Serum (FBS, GIBCO cat# 16140071) supplemented Dulbecco’s Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37°C, 5% CO2. I1 mouse hybridoma cells (ATCC, cat# CRL-2700) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC cat# 30-2003) with 20% FBS at 37°C, 5% CO2.

Protein expression and purification

The constructs used for expression of stabilized MERS-CoV and SARS-CoV-2 S2P trimer proteins were from our previous studies [23]. The mammalian expression plasmid for dimeric soluble ACE2 was purchased from Addgene (#154101). Expi293F cells were used for transient transfection with the suitable S2P stabilized S-expression plasmids or other vectors by using 1 mg/mL polyethylenimine (PEI, Polysciences). The supernatant was harvested and the S trimer was purified using Ni-NTA resin (Smart-Lifesciences) in accordance with the manufacturer's protocol five days after transfection. Prior to use, all proteins were further evaluated for size and purity through SDS-PAGE.

Antibody expression and purification

Monoclonal and bispecific antibodies tested in this study were constructed and produced at Fudan University. For each antibody, variable genes were optimized for human cell expression and synthesized by HuaGene (Shanghai, China). VH and VL were inserted separately into gWiz plasmids that encode the constant region for H chain and L chain. Antibodies were expressed in Expi293F (ThermoFisher, A14527) by co-transfection of H chain and L chain expressing plasmids using polyethylenimine and culture at 37 °C with shaking at 125 rpm and 8% CO2. On day 5 for antibody purification using MabSelectTM PrismA (Cytiva, 17549801) affinity chromatography.

Binding assays by ELISA

100 ng per well of SARS-CoV-2 S trimer or sACE2 protein was coated overnight at 4 °C in a 96-well plate (MaxiSorp Nunc-immuno, Thermo Scientific, USA). The ELISA plates were then blocked with 200 μl blocking buffer (3% BSA) in PBST (0.05% Tween-20 in PBS) at 37 °C for 2 h. Afterwards, purified antibodies were serially diluted using dilution buffer (3% BSA in PBST), incubated at 37 °C for 1 h. Next, 100 μl of 5,000-fold diluted Peroxidase AffiniPure goat anti-human IgG (H + L) antibody (Promega) was added into each well and incubated for 1 h at 37 °C. The plates were washed between each step with PBST in three times. Finally, the TMB substrate (Promega) was added and incubated before the reaction was stopped using 1 M sulfuric acid. Absorbance was measured at 450 nm.

Detecting antibody binding ability by Flow Cytometry

HeLa cells (1 × 10⁶ cells/well) were seeded in six-well plates. After 24 hours, the cells were transfected with plasmids encoding ACE2, MERS-CoV spike, or SARS-CoV-2 spike. Following transfection for 24–48 h, the HeLa cells were digested with 0.25% trypsin and washed twice with PBS. The cells were then resuspended in 1 mL of FACS buffer, and 100 μL of the cell suspension was transferred to a 1.5 mL tube. Parental and bispecific antibodies were added to the corresponding tubes, and the cells were incubated for 30 min at room temperature. After rinsing three times with FACS buffer, the cells were stained with Alexa Fluor 488 AffiniPure Goat Anti-Human IgG (H + L) (33126ES, YEASEN, 0.5 μL/tube) in FACS buffer for 15 min at room temperature. Following staining, the cells were washed and analyzed using a BD LSRFortessa flow cytometer. Data were analyzed using FlowJo software.

Size Exclusion Chromatography

SEC was used to assess physicochemical homogeneity and to resolve monomers from non-monomeric species. Antibodies were analyzed using an AKTA purifier (GE Healthcare). BsAbs (100 μg; 200 μg/mL) was analyzed by SEC on a Superdex S200 10/150 GL column (Cytiva) or Superose™ 6 Increase 10/300 GL column (Cytiva), kept at 4°C temperature, with a mobile phase of PBS and a flow rate of 0.2 mL/min. Purity was confirmed by the predominantly monomeric peaks observed for the bsAbs.

Construction and production of pseudoviruses

Plasmids encoding the MERS-CoV, SARS-CoV, and SARS-CoV-2 variants S were constructed. HEK293 T cells were transfection with the indicated spike gene using Polyethylenimine (Polyscience). Cells were cultured overnight at 37°C with 5% CO2 and VSV-G pseudo-typed ΔG-luciferase (G*ΔG-luciferase, Kerafast) was used to infect the cells in DMEM at a multiplicity of infection of 5 for 4 h before washing the cells with 1×DPBS three times. The next day, the transfection supernatant was collected and clarified by centrifugation at 3000 g for 10 min. Each viral stock was then incubated with 20% I1 hybridoma (anti-VSV-G; ATCC, CRL-2700) supernatant for 1 h at 37 °C to neutralize the contaminating VSV-G pseudotyped ΔG-luciferase virus before measuring titres and making aliquots to be stored at −80 °C.

Pseudovirus neutralization assays

Neutralization assays were performed by incubating pseudoviruses with serial dilutions of antibodies, and scored by the reduction in luciferase gene expression. In brief, Vero E6 cells were seeded in a 96-well plate at a concentration of 2 × 104 cells per well. Pseudoviruses were incubated with serial dilutions of the test samples in triplicate for 30 min at 37 °C. The mixture was added to cultured cells and incubated for an additional 24 h. The luminescence was measured by Luciferase Assay System (Beyotime). IC50 was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC50 values were calculated using nonlinear regression in GraphPad Prism.

Viruses and biosafety

SARS-CoV GZ50 (GenBank accession number AY304495) was an archived clinical isolate at the Department of Microbiology, HKU. MERS-CoV strain EMC/2012 was previously provided by Dr. Ron Fouchier (Erasmus Medical Center) [48]. SARS-CoV-2 WT D614G (GISAID: EPL_ISL_497840), BA.1 (GISAID: EPI_ISL_6841980), XBB.1 (GISAID: EPI_ISL_15602393), EG.5.1 (GISAID: EPI_ISL_18461518) and BA.2.86 (GISAID: EPI_ISL_18986956) strains were isolated from the respiratory tract specimens of laboratory-confirmed COVID-19 patients in Hong Kong [49]. SARS-CoV-2 was cultured using Vero-E6-TMPRSS2. SARS-CoV was cultured in Vero-E6 cells. All the viruses were titrated by plaque assays. All experiments with infectious SARS-CoV-2, SARS-CoV and MERS-CoV were performed according to the approved standard operating procedures of the Biosafety Level 3 facility at the Department of Microbiology, HKU.

Authentic virus neutralization

An end-point dilution assay in a 96-well plate format was performed to measure the neutralization activity of select purified bsAbs as described previously [49]. In brief, each antibody was serially diluted (fivefold dilutions) starting at 50 μg/mL. Triplicates of each antibody dilution were incubated with indicated live virus at a MOI of 0.1 in DMEM with 7.5% inactivated fetal calf serum for 1 h at 37 °C. After incubation, the virus – antibody mixture was transferred onto a monolayer of Vero-E6 cells grown overnight. The cells were incubated with the mixture for 70 h. The cytopathic effects were visually scored for each well in a blinded fashion by two independent observers. The results were then converted into percentage neutralization at a given concentration, and means ± SEM were plotted using a five-parameter dose – response curve in GraphPad Prism.

Supplementary Material

supplementary.docx

Author contributions

P.W. designed and supervised the study; M.L., C.Z., J.S., and X.W. conducted the biological experiments with assistance from G.C., Y.L., and X.Z. M.L., H.C., and P.W. analyzed the results and wrote the manuscript. All authors reviewed, commented, and approved the manuscript.

Disclosure statement

P.W. is listed as an inventor on a patent application for the antibody 2-36 described in this manuscript. All other authors declare no conflict of interest.
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