Current Status and Perspectives of Therapeutic Antibodies Targeting the Spike Protein S2 Subunit against SARS-CoV-2

Abstract

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has devastated public health and the global economy. New variants are continually emerging because of amino acid mutations within the SARS-CoV-2 spike protein. Existing neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) within the spike protein have been shown to have reduced neutralizing activity against these variants. In particular, the recently expanding omicron subvariants BQ 1.1 and XBB are resistant to nAbs approved for emergency use by the United States Food and Drug Administration. Therefore, it is essential to develop broad nAbs to combat emerging variants. In contrast to the massive accumulation of mutations within the RBD, the S2 subunit remains highly conserved among variants. Therefore, nAbs targeting the S2 region may provide effective cross-protection against novel SARS-CoV-2 variants. Here, we provide a detailed summary of nAbs targeting the S2 subunit: the fusion peptide, stem helix, and heptad repeats 1 and 2. In addition, we provide prospects to solve problems such as the weak neutralizing potency of nAbs targeting the S2 subunit.

1. INTRODUCTION

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins form homotrimers on the virus surface and comprises two subunits, S1 and S2¹⁾ (Fig. 1A). The S1 subunit (S_1-685) contains an N-terminal domain (NTD) and a receptor-binding domain (RBD), which mediate the binding of virus and host cell receptor angiotensin-converting enzyme 2 (ACE2).^1,2) The S2 subunit (S_686-1273) contains the fusion peptide (FP), stem helix (SH), and heptad repeats 1 and 2 (HR1 and HR2, respectively) and mediates viral-host membrane fusion.^1–3)

Fig. 1. SARS-CoV-2 Spike Structure and Variant-Related Mutations

(A) Schematic structure of SARS-CoV-2 spike protein. NTD, N-terminal domain; RBD, receptor-binding domain; SD1, subdomain 1; SD2, subdomain 2; S1/S2, furin cleavage site; S2’, S2 sub-cleavage site; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; SH, stem helix; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Amino acid mutations in the S2 subunit of SARS-CoV-2 variants (https://covariants.org). The number of mutations in S1, NTD, RBD, and S2 are shown.

Because of their critical role in mediating viral entry, SARS-CoV-2 spike proteins are major targets for neutralizing antibodies (nAbs).⁴⁾ Since the onset of the COVID-19 pandemic, nAbs against SARS-CoV-2 have been developed and authorized for emergency use, most of which target the S1 subunit, particularly the RBD.^4,5) In fact, the number of SARS-CoV-2 spike nAbs registered on the CoV-AbDab database⁶⁾ exceeds 5000, and the number of nAbs targeting the S2 subunit is approximately 30, which is very limited (accessed on 26 August, 2023).

Anti-RBD antibodies have attenuated neutralizing activity because of amino acid mutations in the RBD region of SARS-CoV-2 variants.^7–9) Developing broad-spectrum nAbs in response to SARS-CoV-2 immune escape mutations is important. Compared to that of the S1 subunit, the amino acid sequence of the S2 subunit is relatively conserved among SARS-CoV-2 variants (Fig. 1B). Previous studies have reported that some antibodies targeting S2 epitopes of spike proteins tend to have broad neutralizing activity.^10–13)

In this review, we discuss the characteristics and functions of SARS-CoV-2 S2 subunit–targeted monoclonal antibodies (mAbs) and provide insights and potential strategies to address the weaknesses of these antibodies.

2. MABS TARGETING THE FP REGION

Proteolytic cleavage at the S2′ site is either processed by enzymes such as the transmembrane protease serine 2 (TMPRSS2) via the plasma pathway or mediated by lysosomal cathepsins during virion endocytosis.¹⁴⁾ Proteolytic cleavage at the S2′ site of the spike protein and outstretch of the FP are required for spike protein–mediated membrane fusion. The sequence of FP is highly conserved among SARS-CoV-2 variants and remains conserved in alpha- and beta-coronaviruses.^15,16) MAbs targeting the FP showed broad binding ability to alpha- and beta-coronavirus spike proteins.¹⁷⁾ Depleting FP-specific antibodies from the serum of COVID-19 convalescent patients reduced SARS-CoV-2 neutralizing activity.¹⁸⁾ FP-specific antibodies isolated from COVID-19 convalescent patients and immunized mice (Table 1) showed broad-spectrum neutralizing activity.

Table 1. Monoclonal Antibodies Targeting Fusion Peptide Region within the SARS-CoV-2 Spike S2 Subunit

Name	Source	Epitope region	Neutralizing potency	IC50 pseudotyped virus (µg/mL)	IC50 authentic virus (µg/mL)	Fc-mediated effector function	Reference
COV44-62	COVID-19 convalescent patient	815-823	β-CoVs: SARS-CoV-2, SARS-CoV, HCoV-OC43, MERS-CoV α-CoVs: HCoV-NL63, HCoV-229E	SARS-CoV-2 WT: 9.80, Alpha: 11.24, Beta: 7.06, Gamma: 12.80, Delta: 13.28, Mu: 6.52, Omicron BA.1: 10.38, BA.2: 20.26, BA.4/5: 51.89	SARS-CoV-2: 66.3, MERS-CoV: 1.07	n.a.	19)
COV44-79		815-823	β-CoVs: SARS-CoV-2, SARS-CoV, HCoV-OC43 α-CoVs: HCoV-NL63, HCoV-229E	SARS-CoV-2 WT: 21.53, Alpha: 24.21, Beta: 19.66, Gamma: 30.64, Delta: 45.33, Mu: 16.51, Omicron BA.1: 33.02, BA.2: 55.44, BA.4/5: 49.81	SARS-CoV-2: 101.2, MERS-CoV: n.d.	n.a.	19)
76E1	COVID-19 convalescent patient	809-833	β-CoVs: SARS-CoV-2, SARS-CoV, HCoV-OC43, MERS-CoV α-CoVs: HCoV-NL63, HCoV-229E	SARS-CoV-2: 0.42, SARS-CoV: 4.82, HCoV-OC43: n.d., MERS-CoV: 3.83, HCoV-NL63: n.d., HCoV-229E: 1.16	SARS-CoV-2: 0.37, SARS-CoV: n.d., HCoV-OC43: 4.66, MERS-CoV: n.d., HCoV-NL63: 3.94, HCoV-229E: 1.60	n.a.	21)
VN01H1	COVID-19 convalescent patient and vaccinated individuals	811-825	β-CoVs: SARS-CoV-2, SARS-CoV, MERS-CoV, WIV-1, PDF-2180 α-CoVs: HCoV-NL63, HCoV-229E	TMPRSS2(−) SARS-CoV-2: n.d., PDF-2180: 2.7 TMPRSS2(+) SARS-CoV-2: 28.8, SARS-CoV: 16.1, MERS-CoV: 31.3, HCoV-NL63: 2.7, HCoV-229E: 2.5, WIV-1: 0.4	Washington-1: 43.0, Omicron BA.1: 12.5, BA.2: 19.7	n.a.	22)
VP12E7		811-825	β-CoVs: SARS-CoV-2, SARS-CoV, MERS-CoV, WIV-1, PDF-2180 α-CoVs: HCoV-NL63, HCoV-229E	TMPRSS2(−) SARS-CoV-2: n.d., PDF-2180: 13.5 TMPRSS2(+) SARS-CoV-2: 113.8, SARS-CoV: 24.1, MERS-CoV: 84.4, HCoV-NL63: 2.7, HCoV-229E: 13.6, WIV-1: 8.0	n.a.	n.a.	22)
C77G12		811-825	β-CoVs: SARS-CoV-2, SARS-CoV, MERS-CoV, WIV-1	TMPRSS2(−) SARS-CoV-2: 3.7, PDF-2180: n.d. TMPRSS2(+) SARS-CoV-2: 7.0, SARS-CoV: 37.6, MERS-CoV: 83.2, HCoV-NL63: n.d., HCoV-229E: n.d., WIV-1: 1.2	Washington-1: 25.9, Omicron BA.1: 2.9, BA.2: 5.4	n.a.	22)
fp.006	COVID-19 convalescent patient	813-825	SARS-CoV-2	SARS-CoV-2 WT: 0.74, Alpha: 15.6, Beta: 29.63, Gamma: 0.41, Delta: 41.98, Omicron BA.1: 19.15, BA.2: n.d., BA.2.75: 80.78, BA.2.75.2: 30.31, BA.4/5: n.d.	n.a.	n.a.	23)

n.d., not determined. n.a., not application.

COV44-62 and COV44-79, FP-targeted mAbs isolated from COVID-19 convalescent patients, showed broad neutralizing activity against alpha- and beta-coronaviruses, including SARS-CoV-2 omicron subvariants. COV44-62 and COV44-79 showed therapeutic activity by inhibiting spike-mediated cell fusion.¹⁹⁾ FP appears partially surface-exposed in a range of coronavirus spike proteins such as SARS-CoV-2.^19,20) However, the binding affinities of these antibodies’ Fabs were higher for the S2 subunit than for the pre-fusion-stabilized spike protein, which suggests that the S1 subunit may partially occlude antibody access to this site on an adjacent protomer.¹⁹⁾

76E1, an FP-targeted mAb isolated from a COVID-19 convalescent patient, showed neutralizing activity against SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron.²¹⁾ The receptor-binding process to ACE2 induces a distinct conformational change in the pre-fusion spike trimer, especially for the exposure of the FP and the S2′ site. Once the FP and S2′ sites are exposed, 76E1 binds to the binding resides and inhibits S2′ cleavage, avoiding the cascade of spike protein rearrangement and blocking virus-host cell membrane fusion and viral entry.²¹⁾ Indeed, hACE2 increased S2′ cleavage by trypsin, and 76E1 inhibited its cleavage.²¹⁾

VN01H1, C77G12, and VP12E7 were FP-targeted mAbs isolated from COVID-19 convalescent and vaccinated individuals. VN01H1 and VP12E7 showed neutralizing activity against beta-coronaviruses, including SARS-CoV-2 and alpha-coronaviruses such as HCoV-NL63.²²⁾ C77G12 showed neutralizing activity only against beta-coronaviruses and showed the highest relative activity against SARS-CoV-2.²²⁾ Furthermore, VN01H1 and C77G12 also inhibited SARS-CoV-2 spike-mediated cell–cell fusion, suggesting that FP-targeted mAbs could prevent spike proteolytic activation or fusogenic rearrangements, thereby inhibiting membrane fusion and viral entry.²²⁾ The addition of ACE2 enhanced the binding of these antibodies to the cell surface-expressed SARS-CoV-2 spike protein, suggesting that receptor binding to ACE2 induces a conformational change that exposes the cryptic FP epitope.^21,22) VN01H1 and C77G12 also showed neutralizing activity against authentic SARS-CoV-2 omicron BA.1 and BA.2, its neutralizing ability was enhanced by a modification to a smaller format such as single-chain variable fragment, suggesting that the smaller size of mAbs can make it more accessible for binding to the epitopes in FP.²²⁾

fp.006, an FP-targeted mAb isolated from COVID-19 convalescent patients, showed neutralizing activity against SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron. Furthermore, fp.006 inhibited SARS-CoV-2 spike-mediated cell–cell fusion.²³⁾

The superimposition of the epitope structures bound by these mAbs onto the pre-fusion spike structure suggested that these mAbs bind to the core epitope with slightly different binding modes, all to the unexposed inner face in the pre-fusion structure.¹⁵⁾ Receptor binding to ACE2 induces a conformational change in the spike structure, exposing the cryptic FP epitope.^21,22) After the receptor-binding process, these mAbs are expected to bind the epitope after the exposure of the FP inner face. Therefore, the combination of FP-targeted mAbs with ACE2 mimics that destabilize the pre-fusion spike has been shown to enhance the therapeutic efficacy of antibodies against SARS-CoV-2.^21,22)

3. MABS TARGETING THE SH REGION

When the trimeric spike protein undergoes a conformational change from the pre-fusion to the post-fusion state, the S2 SH region is elongated to form a six-helix bundle (6-HB).^24–26) As the pre-fusion to post-fusion conformational change of the spike protein is essential for virus-host membrane fusion, mAbs targeting the SH region are expected to block SARS-CoV-2 infection.²⁴⁾ SH-targeted mAbs include those isolated from COVID-19 convalescent patients and isolated from mice immunized with the trimeric spike ectodomain or spike protein-based vaccines of beta-coronavirus (Table 2). Of note, the S2 subunit SH region within residues 1141–1160 of the spike protein²⁴⁾ is conserved among different SARS-CoV-2 variants and is only partially conserved in alpha- and beta-coronaviruses.^15,16) This region may be an ideal antigen for developing broadly nAbs.^24,27,28)

Table 2. Monoclonal Antibodies Targeting Stem Helix Region within the SARS-CoV-2 Spike S2 Subunit

Name	Source	Binding epitope	Neutralizing potency	IC50 pseudotyped virus (µg/mL)	IC50 authentic virus (µg/mL)	Fc-mediated effector function	Reference
S2P6	COVID-19 convalescent patient	1148-1156	β-CoVs: SARS-CoV-2, SARS-CoV, HCoV-OC43, MERS-CoV, PANG/GD	SARS-CoV-2: 1.4, SARS-CoV: 2.4, HCoV-OC43: 1.3, MERS-CoV: 17.1, PANG/GD: 0.02	TMPRSS2(+) SARS-CoV-2: 1.67	ADCC, ADCP	11)
CC40.8	COVID-19 convalescent patient	1142-1159	β-CoVs: SARS-CoV-2, SARS-CoV, SHC014, PANG17, WIV-1	SARS-CoV-2 WT: 12, Alpha: 13, Beta: 13, Gamma: 7, Delta: 6, SARS-CoV: 15, MERS-CoV: n.d., SHC014: 1, PANG17: 14, WIV-1: 6	n.a.	n.a.	12)
CV3-25	COVID-19 convalescent patient	1149-1167	β-CoVs: SARS-CoV-2, SARS-CoV, WIV-1	SARS-CoV-2 WT: 0.32, Alpha: 0.23, Beta: 0.12, Gamma: 0.47, Delta: 0.25, Omicron: 0.18, WIV-1: 0.71	SARS-CoV-2: 0.87 (Vero E6 cells, microneutralization assay)	ADCC, ADCP	13,30)
1249A8	COVID-19 convalescent patient	1147-1158	β-CoVs: SARS-CoV-2, SARS-CoV, MERS-CoV	SARS-CoV-2 D614G: 4.03	SARS-CoV-2 WA-1: 0.69, Delta: 1.54, Omicron: 2.41 SARS-CoV: 0.57, MERS-CoV: 5.83	ADCP	10,33)
S2-4D	Mice immunized with the SARS-CoV-2 spike S2	1144-1156	SARS-CoV-2	n.a.	SARS-CoV-2 WT: 20.1, Alpha: 20.9, Epsilon: 37.1, Gamma: 21.5, Delta: 27.2	n.a.	34)
S2-5D		1144-1156	SARS-CoV-2	n.a.	SARS-CoV-2 WT: 25.8, Alpha: 21.8, Epsilon: 21.2, Gamma: 16.4, Delta: 15.0	n.a.	34)
S2-8D		1144-1156	SARS-CoV-2	n.a.	SARS-CoV-2 WT: 20.8, Alpha: 12.9, Epsilon: 21.0, Gamma: 10.9, Delta: 30.5	n.a.	34)
S2-4A		1144-1156	SARS-CoV-2	n.a.	SARS-CoV-2 WT: 55.7, Alpha: 26.7, Epsilon: 28.9, Gamma: 10.5, Delta: 40.6	n.a.	34)
WS6	Mice immunized with mRNA encoding the SARS-CoV-2 spike	1143-1159	β-CoVs: SARS-CoV-2, SARS-CoV, RaTG13, SHC014, WIV-1	SARS-CoV-2 WA-1: 4.28, Alpha: 6.31, Beta: 9.83, Gamma: 2.46, Delta: 26.5, Mu: 20.3, Omicron: 3.43, SARS-CoV: 1.93, MERS-CoV: n.d., RaTG13: 0.52, SHC014: 0.11, WIV-1: 2.65, HCoV-229E: n.d.	n.a.	n.a.	29)
28D9	Mice immunized with the spike of HCoV-OC43, SARS-CoV, and MERS-CoV	1150-1157	β-CoVs: SARS-CoV-2, SARS-CoV, HCoV-OC43, MERS-CoV	SARS-CoV-2: 45.3, SARS-CoV: 60.5, HCoV-OC43: 64.9, MERS-CoV: 0.13	SARS-CoV-2: n.d., SARS-CoV: n.d., MERS-CoV: 0.93	n.a.	24)
1.6C7		1150-1157	MERS-CoV	SARS-CoV-2: n.d., SARS-CoV: n.d., HCoV-OC43: n.d., MERS-CoV: 0.39	SARS-CoV-2: n.d., SARS-CoV: n.d., MERS-CoV: 0.08	n.a.	24)
B6	Mice immunized with the spike of MERS-CoV and SARS-CoV	1147-1156	β-CoVs: HCoV-OC43, MERS-CoV, HKU4	SARS-CoV-2: n.d., SARS-CoV: n.d., HCoV-OC43: 4.0, MERS-CoV: 1.7, HKU4: 2.4	n.a.	n.a.	27)
IgG22	Mice immunized with the MERS-CoV spike S2	1147-1156	MERS-CoV	n.a.	SARS-CoV-2: n.d., MERS-CoV: 0.12	n.a.	35)

n.d., not determined. n.a., not application.

4. MABS TARGETING SH FROM COVID-19 CONVALESCENT PATIENTS

S2P6, an antibody isolated from a COVID-19 convalescent patient, showed neutralizing activity against SARS-CoV-2 variants, including alpha, beta, gamma, and delta, as well as SARS-CoV, MERS-CoV, HCoV-OC43.¹¹⁾ However, S2P6 could not neutralize the omicron variant.²⁹⁾ S2P6 had weak neutralizing activity in the absence of TMPRSS2, and its interaction with spike proteins was pH-dependent, with higher binding affinity at serological pH than at endosomal pH.¹¹⁾ It suggests that its inhibitory capacity may be reduced when the virus enters the host cell via the endosomal pathway.

CC40.8 was also an antibody isolated from a COVID-19 convalescent patient. CC40.8 showed neutralizing activity against SARS-CoV and different SARS-CoV-2 variants, including alpha, beta, gamma, and delta, inhibiting virus-host membrane fusion.¹²⁾

CV3-25 showed neutralizing activity against authentic SARS-CoV-2³⁰⁾ and pseudotyped SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron.^13,31,32) CV3-25 was highly potent in neutralizing activity and demonstrated therapeutic activity via Fc-mediated effector function and spike protein–mediated membrane fusion inhibition.^30,32)

1249A8 showed neutralizing activity against SARS-CoV-2, SARS-CoV, and MERS-CoV. Furthermore, it showed therapeutic activity against SARS-CoV and SARS-CoV-2 via Fab-mediated neutralization and Fc-mediated effector function.^10,33)

5. MABS TARGETING SH DERIVED FROM IMMUNIZED MICE

S2-4D, S2-5D, S2-8D, and S2-4A were SH-targeted mAbs generated by hybridoma technology from mice immunized with the SARS-CoV-2 S2 subunit. These antibodies exhibited neutralizing activity against SARS-CoV-2 alpha, epsilon, gamma, and delta variants by inhibiting spike protein–mediated membrane fusion.³⁴⁾

WS6 was an SH-targeted mAb isolated from mice immunized with mRNA encoding the SARS-CoV-2 spike protein. WS6 showed neutralizing activity against SARS-CoV-2 alpha, beta, gamma, delta, and omicron variants and bat, civet, and pangolin beta-coronaviruses related to SARS-CoV and SARS-CoV-2.²⁹⁾ WS6 showed neutralizing activity by inhibition of fusion steps post-viral attachment to ACE2.²⁹⁾

In contrast, several SH-targeted mAbs (28D9, 1.6C7, B6, and immunoglobulin G (IgG)22) isolated from immunized mice showed no neutralizing activity against SARS-CoV-2 (excluding pseudotyped SARS-CoV-2 by 28D9). 28D9 and 1.6C7 showed neutralizing activity against MERS-CoV but not against authentic SARS-CoV and SARS-CoV-2.²⁴⁾ B6 could bind to the SARS-CoV-2 and SARS-CoV spike proteins but showed no neutralizing activity.²⁷⁾ IgG22 could bind to the MERS-CoV, SARS-CoV, and SARS-CoV-2 spike proteins but only showed neutralizing activity against MERS-CoV.³⁵⁾ The superimposition of antigen-binding fragments (Fabs) of S2P6 and B6 showed that they bound to a similar epitope in very different orientations. In contrast, the superimposition of Fabs of B6 and IgG22 had very similar structures when their antibodies bound to the epitope.¹⁶⁾ Therefore, it has been suggested that the epitope accessibility and binding orientation of SH-targeted mAbs influence their neutralizing activity.¹⁶⁾

6. MABS TARGETING OTHER REGIONS OF THE S2 SUBUNIT

Most of the anti-S2 antibodies reported in previous studies target the SH and FP regions, with limited reports of mAbs targeting other regions. In particular, mAbs targeting HR1 and HR2 regions have been reported. HR1 and HR2 form the 6-HB and are critical for spike-mediated membrane fusion of SARS-CoV or SARS-CoV-2, making HR1 and HR2 promising therapeutic targets.^36,37) Furthermore, human mAbs targeting HR1 and HR2 regions of SARS-CoV spike protein showed broad neutralizing activity.³⁸⁾ However, mAbs targeting HR1 or HR2 regions of SARS-CoV-2 have not been fully investigated.

hMab5.17, a humanized antibody targeted at the N-terminal end of the HR2 domain, showed neutralizing activity against SARS-CoV and SARS-CoV-2 variants, including alpha, beta, gamma, and delta.³⁹⁾ IC₅₀ values for hMab5.17 were very similar in authentic or pseudotyped SARS-CoV-2 variants.

RAY53, a humanized antibody that targeted the region spanning the HR1/central helix (CH) at the apex of the S2 domain (residues; 980–1006), showed weak neutralizing activity against pseudotyped SARS-CoV-2 Wuhan-Hu-1 and MERS-CoV but not against SARS-CoV-1 and SARS-CoV-2 omicron BA.1.⁴⁰⁾ Furthermore, RAY53 demonstrated therapeutic activity via Fc-mediated effector function in an in vitro assay, and the original murine antibody, 3A3, inhibited spike protein–mediated membrane fusion.⁴⁰⁾

The HR2 region is highly conserved among SARS-CoV-2 variants with approximately 100% (except gamma variants), and the HR1/CH-spanning region (residues; 980–1006) is also highly conserved across all beta-coronaviruses known to infect humans^16,40); however, further studies are needed to identify mAbs targeting these regions as broad-spectrum nAbs against SARS-CoV-2.

7. CONCLUDING REMARKS AND PROSPECTS

Despite the potential advantages, such as broad conservation and functional importance, the S2 subunit is not yet a crucial focus of therapeutic mAb development. The main limitation of the S2-targeted antibodies described in this review is their relatively low in vitro neutralizing activity. RBD-targeted nAbs have reduced neutralizing activity against SARS-CoV-2 variants because of amino acid mutations in the RBD region.^7–9) Therefore, cocktail therapy with mAbs with multiple distinct epitopes has been approved for clinical use by the United States Food and Drug Administration (FDA).⁴¹⁾ However, these cocktail therapies have reduced neutralizing activity against the omicron variant and subvariants,^41–43) and the FDA has restricted the use of these cocktails. Furthermore, the cocktail approach is complicated and increases manufacturing costs and volumes, making it not an ideal strategy to meet the high demand for COVID-19 therapeutics.^44,45) One alternative is the development of multispecific antibodies that have the advantages of cocktails. To date, bispecific antibodies (bsAbs) with epitopes in different regions within the S1 subunit, such as RBD + NTD or RBD + RBD, have enhanced neutralizing activity and spectrum against multiple SARS-CoV-2 variants in comparison with parental antibodies and the cocktails.^46–49) Although most current bsAbs target multiple epitopes within the S1 subunit, bsAbs targeting the RBD and S2 subunits have been reported.

Bi-Nab_35B5-47D10 was an IgG1-like bsAb combining anti-RBD antibody 35B5 and anti-S2 antibody 47D10.⁵⁰⁾ Bi-Nab_35B5-47D10 showed more neutralizing activity against pseudotyped SARS-CoV-2 variants, including alpha, beta, and delta, than the parental mAb. Furthermore, Bi-Nab_35B5-47D10 showed more neutralizing activity against omicron BA.1 and BA.2 than the parental mAb or cocktail.⁵⁰⁾ Therefore, S1 and S2 subunit–targeted bsAbs have the potential to generate multispecific antibodies with broad neutralizing activity and to block viral binding and fusion simultaneously.

Acknowledgments

This study was supported by research grants from the Japan Agency for Medical Research and Development (AMED, 21fk0108568h0001) and JSPS KAKENHI (JP22K15284) to Y.Y.

Conflict of Interest

The authors declare no conflict of interest.

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