Antibodies that can bind to the spike protein of the coronavirus SARS-CoV-2

Abstract

The present disclosure relates to antibodies useful for preventing, treating and / or diagnosing coronavirus infections, including COVID-19, and diseases and / or complications associated with coronavirus infections. In particular, the present disclosure relates to antibodies capable of binding to the spike protein of coronavirus SARS-CoV-2 and uses thereof.

Description

[Technical Field]

[0001] This invention relates to antibodies useful for the prevention, treatment, and / or diagnosis of coronavirus infections, including COVID-19, and diseases and / or complications associated with coronavirus infections. [Background technology]

[0002] Severe acute respiratory syndrome caused by a virus called COVID-19 was first reported in Wuhan, China, in December 2019. The virus spread rapidly worldwide, becoming a pandemic within 12 months with more than 200 million confirmed infections and more than 44 million deaths. The pathogen, SARS-CoV-2, is a beta-coronavirus related to SARS-CoV-1 and MERS coronaviruses, all of which cause severe respiratory syndrome.

[0003] Within months of the identification of SARS-CoV-2 as the pathogen of COVID-19, there has been considerable progress in our understanding of the disease and the virus. Currently, several validated treatments exist, including dexamethasone, tocilizumab, and monoclonal antibodies (mAbs), which have been shown to be effective when used in both prophylactic and therapeutic contexts (Non-Patent Literature 1). Despite these advances, the pandemic is far from under control, and waves of infection continue.

[0004] Coronaviruses possess four structural proteins: the nucleocapsid, envelope, membrane, and spike (S) protein. The spike protein is the most prominent surface protein. The spike protein has an elongated trimer structure and triggers engagement with target cells and fusion of the viral membrane and host membrane. Both SARS-CoV-2 and SARS-CoV-1 spike proteins utilize angiotensin-converting enzyme 2 (ACE2) as a cell surface receptor. ACE2 is expressed in several tissues, including epithelial cells of the upper and lower respiratory tracts.

[0005] The S protein consists of two subunits: S1, which mediates receptor binding, and S2, which is responsible for the fusion of the viral membrane and the host cell membrane. The S protein is a dynamic structure that can transition to a post-fusion state by cleavage between S1 and S2, depending on receptor binding or trypsin treatment. In some SARS-CoV-2 sequences, a furin protease cleavage site is inserted between the S1 and S2 subunits, and mutations in the cleavage site mitigate disease in animal models. The S1 fragment occupies the distal membrane tip of S and can be subdivided into an N-terminal domain (NTD) and a receptor-binding domain (RBD). Both regions are immunogenic, while the RBD contains an interaction surface for ACE2 binding. Although normally packed against the top of S2, the RBD can reorient itself upward to engage with ACE2. Monoclonal antibodies (mAbs) recognize one or both of the “up” and “down” higher-order structures.

[0006] The S protein is relatively conserved between SARS-CoV-2 and SARS-CoV-1 (76%), but the RBD and NTD are less conserved than the S2 domain (90%) (74% and 50%, respectively). Conservation by MERS-CoV and seasonal human coronaviruses is considerably lower (19-21%). Overall, SARS-CoV-2 antibodies show limited cross-reactivity even with SARS-CoV-1.

[0007] S is involved in viral attachment to target cells through the interaction between ACE2 expressed on the cell surface and the S receptor-binding motif (also known as the ACE-2 footprint), which is a 25-amino acid patch at the tip of the receptor-binding domain (RBD) within the S1 fragment of the spike. After attachment, cleavage of S releases S1, which in turn exposes a hydrophobic fusion loop through a large structural change in S2, allowing the virus to fuse with the host cell membrane, releasing the viral genome into the cytoplasm of the host cell and initiating viral replication. Analysis of a large panel of mAbs generated from SARS-CoV-2 infected individuals has revealed mAbs that bind to multiepitopes across S1 and S2. Most mAbs generated against the original SARS-CoV-2 strain can bind to S with high affinity but exhibit little to no neutralizing activity. Genomic surveillance of SARS-CoV-2 has identified thousands of mutations within structural and non-structural proteins. However, towards the end of 2020, a viral variant that rapidly became locally dominant was described, leading to the global spread and naming of the variant of concern (VoC).

[0008] The highly transmissible alpha (B.1.1.7) was first identified in the UK. B.1.1.7 possesses a 9-amino acid change, including N501Y, on the ACE2 interaction surface within the spike protein. Beta (501Y.V2, also known as B.1.351) was first reported in South Africa. Gamma (P.1, 501Y.V2), possessing 10 and 12 amino acid changes, respectively, within the spike protein, were first reported in Brazil. Delta was first reported in India and is now widespread globally, causing outbreaks in several countries. Omicron BA.1 was first reported in South Africa in late November 2021 and has spread worldwide, becoming the dominant variant in many countries and almost completely replacing Delta.

[0009] A series of sublineages of Omicron, including BA.1.1, BA.2, BA.2.12.1, BA.2.75, and BA.4 / 5, have emerged, overcoming preceding strains and becoming regionally or globally dominant. The Omicron S protein has been found to have more than 30 mutations, including 15 substitutions within the RBD, leading to increased transmissibility (Non-Patent Literature 2) and widespread, significant decreases in neutralizing antibody titers (Non-Patent Literature 3).

[0010] Omicron BA.2 was reported almost simultaneously with BA.1. The rate of omicron infections caused by BA.2 is increasing in several countries, and it has become a dominant sublineage in Denmark and India.

[0011] BA.1.1, which contains an additional R346K mutation in the RBD, accounted for approximately 40% of the world's omicron sequences at one point, and approximately 35-60% in the UK and the US (Non-Patent Literature 4), but was quickly surpassed by BA.2. BA.2 contains eight unique S substitutions, including six in the RBD, and lacks the 13 mutations found in BA.1 (Non-Patent Literature 5), and as of August 2022, it is the dominant strain worldwide. Recently, BA.2.12.1 has been identified in multiple countries and caused a large-scale regional epidemic in North America (58% of sequences as of May 25, 2022) (Non-Patent Literature 6).

[0012] Although there is no evidence that it increases disease severity, BA.2 has been shown to have a slight transmissibility advantage over BA.1. In early April 2022, two new omicron strains were reported from Gauteng province in South Africa and named BA.4 and BA.5. BA.4 and BA.5 (which have the same S sequence) became dominant omicron strains in Gauteng and accelerated a new wave of infections in South Africa.

[0013] Since June 2022, BA.4 / 5 (Non-Patent Literature 7), which exhibits higher receptor binding affinity and significantly enhanced escape from antibody reactions, has rapidly spread from South Africa to the rest of the world, becoming a new globally dominant strain, with BA.5 now dominant in many regions. These variants (especially BA.5) now account for the majority of cases sequenced in many countries.

[0014] In early May 2022, a new Omicron sublineage, named BA.2.75, emerged in India. This strain subsequently spread to many countries, including the United Kingdom, the United States, Australia, Germany, and Canada. However, due to incomplete sequencing and significantly reduced scale in many countries, it is difficult to determine the true prevalence of BA.2.75.

[0015] All of these variants contain multiple mutations within S, including RBDs, NTDs, and potentially changes within the Furin cleavage site between S1 and S2. The RBD mutations found in alpha (N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N501Y), and delta (L452R, T478K) are located on or closely adjacent to the ACE2 interaction surface, where the RBD mutations have the potential to modulate the ACE2 interaction and impair neutralizing antibody binding. The increased affinity of the ACE2 interaction is particularly pronounced for alpha, beta, gamma, and delta (7, 19, 19, and 2x, respectively), which may play a role in enhancing viral transmissibility. Omicron contains an unprecedented number of mutations concentrated in the spike (S) gene, with 30 substitutions, 6 deletions, and 3 insertions. Omicron BA.1 (RBD mutations of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H) contains the unique mutations S371L, G446S, and G496S, and some isolates contain R346K (BA.1.1), while BA.2 contains S371F, T376A, D405N, and R408S. BA.3 does not contain the unique mutations associated with BA.1 and BA.2, is similar to BA.1 at the N-terminus, and appears to be a fusion of the two, switching from the G496S mutation to something similar to BA.2 at the C-terminus.

[0016] BA.2.75 contains multiple mutations in the S protein compared to BA.2, including four substitutions in the NTD (W152R, F157L, I210V, G257S) and four substitutions in the RBD: D339H, G446S, N460K, and R493Q.

[0017] Three new variants associated with BA.2, namely BA.2.11, BA.2.12.1, and BA.2.13, have also been detected in multiple countries. These contain single mutations L452R, L452Q, and L452M, respectively, compared to the BA.2 spike receptor-binding domain (RBD) (Figure 29). Of these, BA.2.12.1, first identified in New York, has become dominant in the United States, accounting for approximately 58% of SARS-CoV-2 isolates as of May 25, 2022. L452R is found in the delta and kappa variants, L452Q is found in the lambda variant, but L452M is novel.

[0018] Considering the physicochemical properties of the side chain at residue 452, BA.2.13 is expected to undergo a relatively mild change, with the side chain size increasing from L to M while maintaining hydrophobicity. The L to Q transition in BA.2.12.1 introduces a certain polarity, while BA.2.11 is the most radical, introducing a large basic amino acid from L to R.

[0019] Further variants BA.4 and BA.5, possessing the same S sequence, are thought to have evolved from BA.2. The sequences of BA.4 and BA.5 are highly related to the sequence of BA.2 but contain additional mutations. In particular, residues 69 and 70 of the NTD are deleted (also found in Alpha, BA.1, and BA.3), and two additional substitutions within the RBD: L452R (also found in Delta) and F486V. Finally, BA.4 and BA.5 lack the Q493R change seen in BA.1 and BA.2, reverting to Q493, similar to the Victoria / Wuhan strain. Looking at the RBD, BA.4 and BA.5 construct mutations at all of the aforementioned positions: VoC alpha (N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N501Y), and delta (L452, T478K). The only difference is that in BA.4 and BA.5, the beta and gamma mutations are E484A instead of E484K.

[0020] As of September 2022, a new variant related to BA.4 / 5, named BA.4.6, has emerged and is spreading in the United States, where BA.5 is dominant (as of September 10, 2022, the prevalence was 87.5%, a threefold increase from less than 2% of sequences in early July 2022 to over 6% in mid-August 2022). Compared to BA.4 / 5, BA.4.6 contains two further mutations in the spike protein (S): R346T in the RBD and N658S in the C-terminal domain. The R346K mutation in BA.1.1 reduces serum neutralization compared to BA.1 and impairs the activity of several monoclonal antibodies (mAbs), raising concerns that the R346T mutation may enhance antibody evasion compared to BA.4 / 5 (Non-Patent Literature 5). SARS-CoV-2 detection kits using monoclonal antibodies have also been developed. Examples include lateral flow tests using Innova (SARS-CoV-2 Antigen Rapid Qualitative Test) and Quidel (Sofia 2 SARS Antigen FIA). However, these tests have been reported to be highly inaccurate.

[0021] As of January 2023, further variants such as BQ.1 and XBB have emerged, possessing up to eight additional RBD amino acid substitutions compared to BA.2.

[0022] Structural and functional mapping of a panel of monoclonal antibodies (mAbs) isolated from infectious cases has provided considerable understanding of the antigenicity and neutralization mechanisms of S. Most potent neutralizing antibodies bind to or near the footprint of ACE2 and function by blocking ACE2 interaction, thereby preventing cell attachment and infection. A second site of interaction for potent mAbs is near the N-linked glycan at position N343 exemplified by S309, and these antibodies do not block ACE2 interaction but can play a role in destabilizing the S trimer. A third group of potent mAbs binds to the N-terminal domain of S1, but their mechanism of action remains unknown at present. Another potential RBD epitope of interest is outside the ACE2 footprint, and mAbs binding here are not potent neutralizing agents but can still protect effectively in vivo (Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11).

[0023] Following BA.5, several new trends in the evolution of Omicron were observed: i) the emergence of "second-generation" BA.2 variants (including derivatives of BA.5) - variants with long phylogenetic branches, multiple antigenic mutations, and the absence of genetic intermediates, such as BA.2.75, BJ.1, BS.1, BA.2.10.4, and BA.2.3.20 (Non-Patent Document 12), and ii) accelerated antigenic drift observed in both BA.5 (Non-Patent Document 7) and these second-generation BA.2 lineages, particularly BQ.1 and BA.2.75 (https: / / nextstrain.org / nextclade / sars-cov-2 / 21L). Finally, XBB was generated by recombination between two of these second-generation variants (BJ.1 and BM.1.1.1). Many of these variants show significant convergent evolution at known antigenic RBD residues, and the mutations are in regions that can threaten the binding of neutralizing antibodies, thereby resulting in further escape from protection against infection from previous SARS-CoV-2 infections, including vaccination or previous Omicron infections.

[0024] Currently, several lineages are growing rapidly from within both the BA.2 and BA.5 branches. The most notable is the high degree of convergent evolution, especially at antigen RBD positions such as 346, 444, 446, 452, 460, 486, 490, and 494. These lineages include examples from the BA.4 / 5 branch (naturally containing L452R, F486V, and revertant R493Q), such as BA.4.6 and BF.7 (R346T), BA.4.7 (R34,6S), BQ.1 (K444T, N460K), and BQ.1.1 (R346T, K444T, N460K); and the BA.2.75 branch (naturally containing G446S, N460K, and revertant R493Q), BA.2.75.2 (R346T and F486V), BN.1 (R346T, K356T, F490S). Examples of some other second-generation BA.2 variant strains also exist, such as BJ.1 (alias BA.2.10.1.1; R346T, L368I, V445P, G446S, V483A, and F490V), BA.2.10.4 (G446S, F486P, S494P, and R493Q revertant), BS.1 (BA.2.3.2.1; R346T, L452R, N460K, G476S), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R, and Q493R revertant), and finally the BA.2.75xBJ.1 recombinant, XBB (containing R346T, L368I, V445P, G446S, N460K, F486S, F490S relative to BA.2).

[0025] These second-generation BA.2 variants have become globally dominant. As of December 27, 2022, only BQ.1 accounts for 50% of infections (https: / / cov-spectrum.org / explore / World / AllSamples / Past6M), and XBB.1.5 (XBB.1 + F486P) is rapidly expanding in North America.

[0026] Outside the RBD, convergent evolution is still present, albeit at a lower degree. Many second-generation BA.2 variant lines contain deletions or mutations in the NTD, often similar to those found in the VoC, such as Δ144 in BJ.1 and BA.2.10.4 (previously seen in alpha and BA.1), and NSP12 G671S in BJ.1, BA.2.75, and BA.2.10.4 (previously seen in delta).

[0027] All currently approved SARS-CoV-2 vaccines are designed to induce an antibody (and T-cell) response to S and contain the S sequence found in the original Wuhan strain. Therefore, there are certain concerns as to whether S mutations within VoC could cause immune evasion, leading to vaccine failure or susceptibility to recurrent infection in previously infected individuals.

[0028] The extensive mutagenesis of Omicron S inhibits the activity of most mAbs that bind to the three potent antibody binding sites mentioned above, the ACE-2 footprint, around the N343 glycan, and to NTDs. This significantly reduces or completely eliminates the serum's neutralizing capacity from natural infection or vaccination, which contributes to the increased transmissibility and explosive spread of Omicron. [Prior art documents] [Non-patent literature]

[0029] [Non-Patent Document 1] Baum et al.,2020,Science 369,1014-1018 [Non-Patent Document 2] Suzuki et al.,2022“Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.”Nature 603,700-705 [Non-Patent Document 3] Dejnirattisai et al., 2022 “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell 185, 467 - 484 e415 [Non-Patent Document 4] Iketani et al., 2022 “Antibody evasion properties of SARS-CoV-2 Omicron sublineages.” Nature 604, 553 - 556 [Non-Patent Document 5] Nutalai et al., 2022 [Non-Patent Document 6] Del Rio and Malani, 2022, “COVID-19 in 2022 - The Beginning of the End or the End of the Beginning?” JAMA 327, 2389 - 2390 [Non-Patent Document 7] Tuekprakhon et al., 2022 “Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.” Cell 185, 2422 - 2433 e2413 [Non-Patent Document 8] Huo et al., 2020 [Non-Patent Document 9] Sun et al., 2021 [Non-Patent Document 10] Yuan et al., 2020 [Non-Patent Document 11] Zhou et al., 2020 [Non-Patent Document 12] van der Straten et al. 2022. Immunity 55, 1725 - 1731 [Summary of the Invention] [Problems to be Solved by the Invention]

[0030] The object of the present invention is to identify further improved antibodies useful for preventing, treating and / or diagnosing coronavirus infection and related diseases and / or complications, including COVID-19, particularly the Omicron variant of concern (VoC), and variants of SARS-CoV-2 that have further mutations in the ACE-2 footprint, RBD, and / or NTD within the spike protein, which have yet to be identified. [Means for solving the problem]

[0031] The inventors identified 28 human monoclonal antibodies (mAbs) that recognize the SARS-CoV-2 spike protein (see Table 3). These antibodies showed potent neutralizing activity against SARS-CoV-2. Some of the antibodies in Table 3 showed potent neutralizing effects that were broadly effective against the hCoV-19 / Wuhan / WIV04 / 2019 strain and various lineages of SARS-CoV-2 strains, such as Victoria (Wuhan+S247R), Alpha, Beta, Gamma, Delta, and Omicron (including Omicron BA.2.11, Omicron BA.2.12.1, Omicron BA.2.13, Omicron BA.2.3.20, Omicron BA.2.10.4, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.2.75, BA.2.75.2, Omicron BA.3, Omicron BA.4.6, Omicron BA.4 / 5, Omicron BJ.1, Omicron BS.1, Omicron BN.1, Omicron XBB and / or Omicron XBB.1).

[0032] Many of the mAbs in Table 3 were derived from public V genes (V genes shared by the majority of populations). We previously demonstrated that it is possible to generate further antibodies by swapping the light and heavy chains of the antibodies in Tables 1, 2, and 3, which are derived from the same public V gene. Antibodies derived from the same public V gene provided particularly useful mixed-chain antibodies.

[0033] In particular, the inventors found that antibodies Omi02, Omi03, Omi12, Omi18, Omi28, Omi39, and Omi42 are especially effective in cross-neutralizing SARS-CoV-2 strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron.

[0034] Therefore, the present invention provides an antibody that can bind to the spike protein of the coronavirus SARS-CoV-2, and comprises at least three CDRs of any one of the 28 antibodies listed in Table 3.

[0035] The present invention provides an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising at least three CDRs of either antibody Omi12 or one of the 27 antibodies listed in Table 3.

[0036] The present invention also provides antibody combinations comprising two or more antibodies according to the present invention.

[0037] The present invention also provides combinations of antibodies comprising (a) the antibody of the present invention and (b) an antibody comprising at least three CDRs of the antibodies in Table 1 or Table 2. For example, the antibody may comprise (i) at least four, five, or six CDRs of the antibodies in Table 1 or Table 2; (ii) a heavy chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity with respect to the heavy chain variable domain of the antibody in Table 1 or Table 2; (iii) a light chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity with respect to the light chain variable domain of the antibody in Table 1 or Table 2; and / or (iv) a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having at least 80% identity with respect to the heavy chain variable domain and light chain domain of the antibody in Table 1 or Table 2, respectively.

[0038] The present invention also provides one or more polynucleotides encoding the antibody of the present invention, one or more vectors containing the polynucleotides, or host cells containing the vectors.

[0039] The present invention also provides a method for producing an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising culturing the host cells of the present invention and isolating the antibody from the culture.

[0040] The present invention also provides a pharmaceutical composition comprising (a) an antibody or combination of antibodies of the present invention and (b) at least one pharmaceutically acceptable diluent or carrier.

[0041] The present invention also provides antibodies, combinations of antibodies, or pharmaceutical compositions for use in methods of treating the body of a human or animal by therapy.

[0042] The present invention also provides antibodies, combinations of antibodies, or pharmaceutical compositions for use in methods of treating or preventing coronavirus infection or diseases or complications associated with coronavirus infection.

[0043] The present invention also provides a method for treating or preventing coronavirus infection or a disease or complication related to coronavirus infection in a subject, comprising administering a therapeutically effective amount of the antibody, combination of antibodies, or pharmaceutical composition of the present invention to the subject.

[0044] The present invention also provides a method for identifying the presence of coronavirus or its protein fragments in a sample, comprising (i) contacting the sample with the antibody or combination of antibodies of the present invention, and (ii) detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates the presence of coronavirus or its fragments in the sample.

[0045] The present invention also provides a method for treating or preventing coronavirus infection or related diseases or complications in a subject, comprising identifying the presence of coronavirus according to the method of the present invention, and treating the subject with an antibody or combination, antiviral agent or anti-inflammatory agent according to the present invention.

[0046] The present invention also provides the use of antibodies, combinations of antibodies, or pharmaceutical compositions of the present invention for the prevention, treatment, and / or diagnosis of coronavirus infection or related diseases or complications.

[0047] The present invention also provides the use of antibodies, combinations of antibodies, or pharmaceutical compositions of the present invention for the manufacture of agents for treating or preventing coronavirus infection or related diseases or complications. [Brief explanation of the drawing]

[0048] [Figure 1] Generation of Omicron BA.2 sublineages and Omicron mAb panels. Figure 1 relates only to the first 22 Omicron antibodies (i.e., Omi02-Omi35) disclosed in Tables 13 and 14. (A) FRNT50 titers against Victoria and Omicron BA.1 from donors for Omicron mAb production are shown. (B) FACS plot showing B cell classification using full-length Omicron S. (C) Percentage of RBD and NTD-binding antibodies found in Omicron mAbs compared to early pandemic mAbs. (D) Use of heavy and light chain variable genes. (E) Esomatic mutations found in potent Omicron mAbs (FRNT50 < 100 ng / ml) compared to early pandemic sets. [Figure 2] Neutralization curves using Omicron mAbs. (A) Victoria, Alpha, Beta, Gamma, Delta, and Omicron BA.1 viruses. (B) Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses by Omicron mAbs. (C) Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses by antibodies developed for commercial use. [Figure 3]Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses. (A) Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses 28 days after the second and third administrations of AZD1222 (n=41) and (B) BNT162b2 (n=20). (C) Live virus neutralization assays with Victoria, alpha, beta, gamma, delta, and omicron viruses using serum obtained between 14 and 21 days after symptom onset, and (D) Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses with early and late serum. Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon's matched-pair signed-rank test (A and B) and Mann-Whitney U test (C and D) for analysis. [Figure 4] Pseudoviral neutralization curves. Pseudoviral neutralization curves for BA.1, BA.1.1, BA.2, and BA.3 in mAb(B) beta mAb during the early stages of the pandemic. [Figure 5] Figure 3 shows the neutralizing titers of the viruses for (A) live viruses and (B) pseudoviruses. Geometric mean titers are shown above each column. Wilcoxon's matched-pair signed-rank test was used to calculate the two-tailed p-values. (C) Pseudovirus neutralization curves for selected VH1-58mAb and control VH3-53mAb222 against Victoria and Iota (S477N). [Figure 6] Structure of BA.1 RBD with Omi-12Fab. (A) Two ternary complexes of Omi-12 and beta-54 Fab and BA.1 in crystalline asymmetric units (generated by fitting high-resolution structures of BA.1 RBD, Omi-12, and beta-54 to a low-resolution ternary complex density) are compared by superimposing the RBDs. The Fab of one complex is brightly colored (HC is red, LC is blue in the schematic), and the Fab of the other complex is palely colored. (B) Binding modes of Omi-12. (C) Magnified view of the difference in binding of Omi-12 with Fab253 compounded with an early pandemic RBD (pale blue) and beta-47 compounded with a beta RBD (pale cyan). (D) The somatic mutation V53P contributes to the refolding of the H3 loop, allowing Q493R to be accommodated in Omi-12. [Figure 7] Pseudoviral neutralization assays of BA.4 / 5 with vaccine and BA.1 immunized serum. IC50 values ​​of the indicated viruses were obtained using serum from vaccinated individuals 28 days after the third dose of vaccine (A) AstraZeneca AZD AZD1222 (n=41) and 4 weeks after the third dose of vaccine (B) Pfizer BNT162b2 (n=20). Serum was collected from breakthrough BA.1 infected volunteers at (C) early stage, ≤14 days (n=12) (median 13 days) from symptom onset, and (D) late stage, ≥21 days (median 38 days) from symptom onset, n=16. Neutralization titers were compared against Victoria (early pandemic strain), BA.1, BA.1.1, BA.2, and BA.3. Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon's matched-pair signed-rank test for analysis. [Figure 8] Pseudoviral neutralization assays against omicron and commercially available monoclonal antibodies. Neutralization curves of a panel of 28 monoclonal antibodies prepared from samples taken from vaccinated individuals infected with BA.1. The titration curve for BA.1 is compared to that of BA.1, BA.1.1, BA.2, and BA.3. mAbs presumed to be affected by L452R and F486L are shown. [Figure 9] Omicron sublineage compared to BA.4 / 5. (A) Comparison of S protein mutations in Omicron BA.1, BA.1.1, BA.2, BA.3, and BA.4 / 5, showing NTD and RBD boundaries. (B) Location of RBD mutations (gray surface, ACE2 footprint in dark green). Mutations common to all Omicron lineages are shown in white (Q493R, which reverted in BA.4 / 5, is shown with an ×), mutations common to BA.1 and BA.1.1 are shown in cyan, mutations specific to BA.1.1 are shown in blue, and mutations specific to BA.2 are shown in magenta. Residue 371 (yellow) is mutated in all Omicron viruses, but differs between BA.1 and BA.2. N343 glycan is shown as a transparent rod. [Figure 10]Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs. (A) Binding of BA.4 / 5 RBD was significantly reduced compared to binding of BA.2, and the binding shown by a single injection of 200 nM RBD into a sample flow cell containing IgG Omi-31 could not be accurately measured. (B~C; E~I) Sensorgrams showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red: original binding curve, black: fitted curve). Dynamical data are also shown. (D) Measurement of affinity between BA.4 / 5 RBD and Omi-12 using 1:1 binding equilibrium analysis. [Figure 11] Interactions between mAbs and BA.4 / 5 mutation sites. Overall structures (left panel) and interactions (≤4Å) (right panel) of the complexes of (A) BA.1-RBD / Omi-31 (PDB 7ZFB), (B) BA.1-RBD / Omi-32 (PDB 7ZFE), (C) BA.1-RBD / Omi-25 (PDB 7ZFD), (D) BA.1-RBD / Omi-42 (PDB 7ZR7), (E) Wuhan-RBD / AZD8895 (PDB 7L7D), and (F) BA.1-RBD / Omi-3 (PDB 7ZF3). In the left panel, RBD is shown as a surface representation, the BA.4 / 5 mutation site is highlighted in magenta, two additional mutation sites at 452 and 486 of BA.4 / 5 are highlighted in cyan, FabLC is shown with a blue ribbon, and HC with a red ribbon. In the right panel, the side chains of RBD, Fab HC, and LC are depicted with gray, red, and blue bars, respectively. In (B), L452R (green bar) is modeled to show that a salt bridge to D99 of CDR-H3 may be formed (yellow dashed line). (D) The beta-RBD / Omi-42 complex shows that Fab is not in contact with either of the two BA.4 / 5 mutation sites. [Figure 12]ACE2 RBD affinity. (A)-(D) SPR sensor gram comparing ACE2 binding of BA.4 / 5 RBD (A) to binding to ancestral (Wuhan) (B), BA.1 (C), and BA.2 RBD (D). Data for Wuhan, BA.1, and BA.2 were previously reported (Nutalai et al., 2022). (E)-(G) Electrostatic surface, (E) from left to right: early pandemic, delta, BA.1 RBD, (F) open view of BA.2 RBD and ACE2 in the BA.2 RBD / ACE2 complex (PDB7ZF7), and (G) BA.4 / 5 RBD (modeled based on the structure of BA.2 RBD). Rhombus shapes in ACE2 and RBD indicate interaction regions. [Figure 13] Antigen Mapping. (A)(B) Neutralization data and model (log titer value) used to calculate the antigen map. Columns represent serum collected from vaccinated volunteers or infected patients. Rows are challenge strains: Victoria, Alpha, Delta, Beta, Gamma, BA.1, BA1.1, BA.2, BA.3, BA.4 / 5, in order. Values ​​are color-coded according to their deviation from the reference value, which is calculated based on serotype as the average of neutralizing titers from the row giving the highest value. (B) Orthogonal plot of the antigen map showing BA.4 / 5 in relation to previous VoCs and the positions of BA.1, BA.1.1, BA.1 and BA.2, calculated from pseudoviral neutralization data. The distance between two positions is proportional to the decrease in neutralizing titer when one of the corresponding strains is challenged with serum obtained by infection by the other. Figure 6. ACE2 / RBD Affinity and Antigen Mapping [Figure 14] Neutralization curves for VH1-58mAb. Pseudoviral neutralization curves for early pandemic mAb253 (Dejnirattisai et al., 2021a) and beta-47 (Liu et al., 2021b) against panels of Victorian and Omicron lineage constructs. [Figure 15]Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs. (A-F) Sensorograms showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red: original binding curve, black: fitted curve). Dynamical data are also shown. (G-K) Binding of BA.4 / 5 RBD was significantly reduced compared to binding of BA.2, and the binding shown could not be accurately measured by injecting 200 nM RBD once into a sample flow cell containing the indicated mAbs. [Figure 16] Sequence changes of BA.2.75 compared with other Omicron sublines. (A) Sequence alignment of BA.2.75 with Omicron sublines Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 / 5. NTD and RBD boundaries are marked. (B) Surface representation of mutant residues in BA.2.75 RBD compared with BA.2 RBD. The location of the BA.2 RBD mutation (gray surface, ACE2 footprint in dark green) is shown, and the mutated residues in BA.2.75 are shown in orange and labeled. [Figure 17] Pseudoviral neutralization assays of BA.2.75 with vaccine and BA.1 and BA.2 immunized serum. (A) IC50 values ​​of the indicated viruses using serum obtained from vaccinated individuals 28 days after the third dose of vaccine Pfizer BNT162b2 (n=22). (B) AstraZeneca AZD AZD1222 (n=41). (C, D) Serum from volunteers infected with vaccine breakthrough BA.1 (n=16) or BA.2 (n=23). (EC) IC50 values ​​of single RBD point mutations inserted into the BA.2 pseudovirus using Pfizer BNT162b2 serum (n=22). Geometric mean titers are shown above each column. Two-tailed P-values ​​were calculated using Wilcoxon's matched pair signed-rank test for analysis. [Figure 18]ACE2 / RBD affinity. SPR sensorgrams showing ACE2 binding of BA.2.75 RBD using ACE2-Fc (A) or biotinylated ACE2 (B) as a ligand, compared to the binding of BA.2 (C), BA.4 / 5 (D), Alpha (E), and BA.2+R493Q (F) to RBD. Data for BA.2, BA.4 / 5, and Alpha have been previously reported by Nutalai et al., 2022, Tuekprakhon et al., 2022, and Dejnirattisai et al., 2022, respectively. [Figure 19] Pseudoviral neutralization assays against monoclonal antibodies. (A) Neutralization curves of a panel of 28 mAbs prepared from samples taken from vaccinated individuals infected with BA.1. The titration curve for BA.2.75 is compared with that of Victoria, BA.1, BA.1.1, BA.2, and BA.4 / 5. IC50 titers are shown in Table 22. (B) Pseudoviral neutralization assays with mAbs developed for human use. IC50 titers are shown in Table 23. Data for Victoria, BA.1, BA.1.1, BA.2, and BA.4 / 5 were used for comparison and were obtained from Tuekprakhon et al., 2022. [Figure 20] Structure of the BA.2.75 RBD / ACE2 complex. (A) Overall structure of the BA.2.75 RBD / ACE2 complex. ACE2 is shown with a green ribbon, RBD is shown on the surface, common mutations in BA.2 are highlighted in magenta, and different mutations are highlighted in orange. (B) Interface of BA.2.75 RBD (gray) and ACE2 (green) compared to the interface of BA.2 and ACE2 (both salmon color). The magnified view shows the interaction between Q496R and Q493 (R493 in BA.2) and ACE2. [Figure 21]Interaction between mAb and BA.75 mutation sites. (A) Front and back views of the binding modes of Omi-3 (PDB, 7ZF3) and Omi-18 (PDB, 7ZFC) compounded with Omicron BA.1 RBD by superimposing RBDs. RBDs are shown as gray surface representations, mutations common to both BA.2 and BA.2.75 are shown in magenta, and four mutations different between the two are shown in cyan. VH and VL are shown as ribbons, with Omi-3 shown in red and blue, and Omi-18 in cyan and salmon colors, respectively. (B) Interaction between N460 of the RBD and CDR-H2 of the Fab. (C) Contact between R493 of the RBD and CDR-H3 of the Fab. In (B) and (C), the RBD associated with Omi-3 is shown in gray, Omi-18 in cyan, and the Fab's color is the same as in (A). (D)AZD1061 is bound to the ancestral SARS-CoV-2 RBD (PDB, 7L7E), with G446 of (E)RBD and CDR-L2 of Fab in contact. (E)AZD8895 is bound to the ancestral SARS-CoV-2 spike RBD (PDB, 7L7E), with Q493 of (F)RBD and CDR-H2 of Fab in contact. In (D) to (F), the RBDs are depicted and color-coded in the same way as in (A), with HC shown in red and LC in blue. [Figure 22] Antigen mapping. (A) An orthogonal plot of the antigen map showing BA.2.75 relative to the previous VoC and the positions of BA.1, BA.1.1, BA.1, and BA.2, calculated from pseudoviral neutralization data. The distance between two positions is proportional to the decrease in neutralizing titer when one of the corresponding strains is challenged with serum obtained by infection by the other. Since the figure is a projection of the three-dimensional distribution, a scale is not provided, but the variation can be calibrated by comparing (i) BA.1 to BA.2 (2.93x decrease) and (ii) BA.2 to BA.4 / 5 (3.03x decrease). (B) Similar to (A), but includes only the Omicron sublineage and early pandemic viruses to allow for a more accurate three-dimensional projection of this subset. Note that the response of these viruses to all serums is included in the calculation. [Figure 23]Pseudoviral neutralization assay against monoclonal antibodies. (A) Neutralization curves of a panel of 28 monoclonal antibodies prepared from samples taken from vaccinated individuals infected with BA.1. The titration curves of the single mutant BA.2.75 in the BA.2 skeleton are compared with those of BA.2 and BA.2.75. IC50 titers are shown in Table 24. [Figure 24] Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.2.75 RBD and selected mAbs. (A) Binding of Omi-29 (IGHV3-53) to BA.2.75 RBD is significantly reduced compared to that of BA.2, as shown by a single injection of 1 μM Omi-29Fab into a sample flow cell containing biotinylated BA.2 or BA.2.75 RBD. (B) Binding of Omi-36 (IGHV3-66) to BA.2.75 RBD is significantly reduced compared to that of BA.2, as shown by a single injection of 0.2 μM BA.2 or BA.2.75 RBD into a sample flow cell containing IgG-type Omi-36. (C-H) Sensorgrams showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red / colored: original binding curve, black: fitted curve). Dynamical data are also shown. [Figure 25] Neutralization of BA.2.75 by panel of convalescent serum collected from infections with past variants. Shown serum neutralizing titers against BA.2.75 and the indicated pseudovirus. Data other than BA.2.75 were obtained from Tuekprakhon et al., 2022. [Figure 26] Primers for site-directed PCR mutagenesis of BA.2.75 RBD. Site-directed PCR mutagenesis was performed using the BA.2 spike construct as a template. D339H, G446S, N460K, and R493Q mutations were introduced using the indicated primers. [Figure 27]Characterization of BA.2.11, BA.2.12.1, and BA.2.13 by pseudoviral neutralization assay, surface plasmon resonance, and structural analysis. (a), (b) IC50 values ​​of the indicated viruses using serum obtained 4 weeks after the third dose of vaccine (a) AstraZeneca AZD1222 (n=41), (b) Pfizer BNT162b2 (n=18). (c) Serum neutralizing titers were measured from vaccinated volunteers who contracted breakthrough BA.1 infection. Comparisons were made with neutralizing titers against Victoria, BA.1, BA.1.1, BA.2, and BA.4 / 5 previously reported in Tuekprakhon et al. (2022). Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon's matched pair signed-rank test for analysis. (d~g) SPR sensorgrams showing ACE2 binding of RBDs of BA.2.11(e), BA.2.12.1(f), and BA.2.13(g) compared to binding to BA.2 RBD(h) (red: experimental binding curve, black: fitted curve). Dynamical data are also shown. Data for BA.2 RBD have been reported by Nutalai et al. (2022). (h~m) Crystal structure of the BA.2.12.1 RBD / beta-27 / NbC1 complex. (h) The overall structure is shown as Cα traces with RBD (gray), beta-27HC (red), and LC (blue) and NbC1 (yellow). The Cα of residues L452Q, F486, and Q493R (L, F, and R in BA.2, and R, V, and Q in BA.4 / 5) are shown as spheres. (i) Comparison of beta-27 binding modes in the BA.2.12.1 RBD / beta-27 / NbC1 (RBD as surface representation, HC as red, LC as blue), BA.4 / 5 RBD / beta-27 / NbC1 (cyan, PDB 7ZXU), and betaRBD / beta-27 (green, PDB 7PS1) complexes by superimposing RBDs. Apart from the flexible N and C-terminal regions of RBD, significant differences occur between the N-terminus of the Fab HC, α2 helix, 371-375 loop, and G446 loop of RBD and CDR-H1. CDR-L3 has a dual structure in the BA.4 / 5 RBD complex and a single structure in the other two complexes (i).The HCN terminus and CDR-H1 in contact with residue 486 of the RBD differ from those in both the beta and BA.4 / 5 RBD complexes, the latter containing the F486V mutation. This difference is thought to be caused by contact from the C1 nanobody related to symmetry, shown as the gray bond in (j). (k) Structural differences in the G446 loop of the BA.4 / 5 RBD are also induced by crystal contact. (l) The 371-375 loops with the S371F, S373P, and S375F mutations in BA.2.12.1 and BA.4 / 5 RBDs are stabilized by interaction with the CDR-H3 of NbC1. (m) Superposition of BA.2.12.1 (gray), BA.2 (green, PDB 7ZF9), and BA.4 / 5 (cyan) RBDs. (n) The mutation at 452 does not cause significant local structural changes. R452 of BA.4 / 5 has a double structure. [Figure 28] Vaccine-mediated pseudoviral neutralization assay of BA.4.6. BA.1, BA.2, and BA.4.5 immune sera (a-d) and monoclonal antibodies (e-f). IC50 values ​​of the indicated viruses using sera obtained from vaccinated individuals 28 days after the third dose of Pfizer BNT162b2 vaccine (n=22, a). IC50 values ​​of the indicated viruses for sera from volunteers infected with vaccine breakthrough BA.1 (n=14, b), BA.2 (n=23, c), and BA.4 / 5 (n=11, d). Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon's matched-pair signed-rank test for analysis. The neutralization curves for BA.4.6 of a panel of 28 monoclonal antibodies prepared from samples taken from vaccinated individuals infected with BA.1(e) were compared to those for Victoria, BA.1, BA.1.1, BA.2, BA.4 / 5, and BA.2.75 variants. The neutralization curves (e) of a panel of 14 commercially available monoclonal antibodies against the same variants are shown. IC50 values ​​are shown in Tables 29A and 29B. [Figure 29]Pseudoviral neutralization assay. Pseudoviral neutralization assay against omicron monoclonal antibodies for Table 26, which shows IC50 titers. Neutralization curves for a panel of 27 monoclonal antibodies prepared from samples taken from vaccinated individuals infected with BA.1. Titer curves for BA.2.11, BA.2.12.1, and BA.2.13 are compared with those for BA.2. [Figure 30] Surface plasmon resonance (SPR) analysis of the interaction between BA.2.12.1 or BA.2 RBD and selected mAbs (Omi-6 and Omi-31). (a) Measurement of the affinity of BA.2.12.1 RBD to Omi-6 using 1:1 coupled equilibrium analysis. (b), (c), (d) Sensorograms showing the interaction between BA.2.12.1 or BA.2 RBD and selected mAbs (red: original coupling curve, black: fitted curve). Dynamical data are also shown. [Figure 31] Neutralization assay. Neutralization curves using lentiviruses pseudotyped with the S genes of the BA.2 subline (A) Omi-mAb and (B) commercially available mAb. See also Table 32. The "BA.4+all" variant is a synthetic variant designed according to the evaluation of different mutations occurring in the SARS-CoV-2 omicron S gene. These mutations were combined and incorporated into the omicron BA.4S gene to produce an artificial S gene called "BA.4+all". This variant was created solely as an experimental tool, does not exist in nature, and does not correspond to the S gene of any circulating SARs-CoV-2 variant. [Figure 32] Serum neutralizing IC50 titers (dilution factor) of lentivirus pseudotyped with the S gene of the indicated BA.2 sublineage. (A) Serum obtained 28 days after the third dose of BNT162b2 vaccine, or (B) BA.1, (C) BA.2, or (D) BA.4 / 5 infection. Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon's matched-pair signed-rank test (C and D) and Mann-Whitney U test (E). [Figure 33]Antibody binding heatmap. A heatmap showing the IC50 (μg / ml) of various antibodies against Victoria and Beta strains in both vaccinated and unvaccinated samples. [Figure 34] Neutralization assay. Neutralization curve using lentivirus pseudotyped with the S gene of the BA.2 sublineage shown. [Figure 35] Heatmap of IC50 neutralization titers of a panel of BA.1(Omi)mAbs. Pseudoviral neutralization IC50 titers of the shown mAbs against a panel of pseudoviruses expressing variant S sequences. Live viral IC50 values ​​for variants seen in the early stages of the pandemic are also included for comparison. Live viral assay data and pseudoviral data for Victoria, BA.2, and BA.4 / 5 were previously reported by Tuekprakon et al. (2022). [Modes for carrying out the invention]

[0049] The antibody of the present invention The antibody of the present invention specifically binds to the spike protein of SAR-CoV-2. In particular, it specifically binds to the S1 subunit of the spike protein, such as the receptor-binding domain (RBD) or the N-terminal domain (NTD).

[0050] The antibody of the present invention may contain at least three CDRs of the antibodies in Table 3. The antibody may contain at least four, five, or all six CDRs of the antibodies in Table 3. The antibody may contain an amino acid sequence having at least 80% sequence identity with the heavy chain variable domain of the antibodies in Table 3, or a heavy chain variable domain consisting thereof. The antibody may contain an amino acid sequence having at least 80% sequence identity with the light chain variable domain of the antibodies in Table 3, or a light chain variable domain consisting thereof. The antibody may contain an amino acid sequence having at least 80% identity with the heavy chain variable domain and light chain variable domain of the antibodies in Table 3, or a heavy chain variable domain and a light chain variable domain consisting thereof. The antibody may be any one of the antibodies in Table 3.

[0051] Table 3 lists 28 individual antibodies identified from recovered Breakthrough Omicron SARS-CoV-2 infected patients who had already received two doses of the Pfizer vaccine. Table 1 lists 42 individual antibodies previously identified from recovered COVID-19 patients [Dejnirattisai, Wanwisa, et al. "The antigenic anatomy of SARS-CoV-2 receptor binding domain." Cell 184(8)(2021):2183-2200; Supasa, Piyada, et al. "Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera." Cell 184(8)(2021):2201-2211; Zhou, Daming, et al. "Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera." Cell 184(9)(2021):2348-2361; Dejnirattisai, Wanwisa, et al. "Antibody evasion by the P.1 strain of SARS-CoV-2.Cell 184(11)(2021):2939-2954; Liu, Chang, et al. "Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum."Cell 184(16)(2021):4220-4236.]. Table 2 lists 28 individual antibodies previously identified from recovered beta-SARS-CoV-2 infected patients [Liu, C et al. "The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants".Cell hostµbe 30(1)(2021):53-68]. The antibodies in Table 1 are also referred to herein with the prefix "COVOX," for example, as COVOX-222.The antibodies in Table 2 are also referred to as "β50," for example, with the prefix "β." The antibodies in Table 3 are also referred to as "O02," for example, with the prefix "O." Tables 1-3 list the sequence numbers for the nucleotide and amino acid sequences of the heavy chain variable region and light chain variable region, as well as the complementarity-determining region (CDR) of the variable chain of each antibody.

[0052] The antibodies listed in Table 3 may be selected from the following group: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36, and Omi38. Surprisingly, these antibodies were found to retain potent neutralizing activity against live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron (e.g., IC50 ≤ 0.1 μg / ml for all live strains tested).

[0053] The antibodies listed in Table 3 may be selected from the following group: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, and Omi08. Surprisingly, these antibodies were found to retain potent neutralizing activity against live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron (e.g., IC50 ≤ 0.05 μg / ml for all live strains tested).

[0054] The antibodies in Table 3 can be selected from the following group: Omi03, Omi12, Omi02, Omi39, and Omi42. Surprisingly, these antibodies were found to retain potent neutralizing activity against live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron (e.g., IC50 ≤ 0.02 μg / ml for all live strains tested).

[0055] The antibodies in Table 3 can be selected from the following groups: Omi03 and Omi12. Surprisingly, these antibodies were found to retain very potent neutralizing activity against live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron (e.g., IC50 ≤ 0.01 μg / ml for all live strains tested).

[0056] The antibodies in Table 3 can be selected from the following group: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39, and Omi42. Surprisingly, these antibodies were found to retain very potent neutralizing activity against live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron.

[0057] Therefore, in one embodiment, the antibody in Table 3 may be Omi03. Omi03 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 695, 696, and 697, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 698, 699, and 700, respectively. In one embodiment, the antibody of the present invention may contain or include a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 692). In one embodiment, the antibody of the present invention may contain or include a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 694). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi03 (i.e., SEQ ID NOs. 692 and 694, respectively), respectively.

[0058] The heavy chain domain of Omi03 originates from the IGHV3-53 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi03 but may not contain the light chain of Omi03. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 695, 696, and 697, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi03 (i.e., SEQ ID NO. 692). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 692.

[0059] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi03 but may not contain the heavy chain of Omi03. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 698, 699, and 700, respectively. The antibody may contain or comprise a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi03 (i.e., SEQ ID NO. 694). The antibody may contain or comprise a light chain variable domain containing SEQ ID NO. 694.

[0060] In one embodiment, the antibodies in Table 3 may be Omi12. Omi12 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 735, 736, and 737, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 738, 739, and 740, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi12 (i.e., SEQ ID NO: 732). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi12 (i.e., SEQ ID NO: 734). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi12 (i.e., SEQ ID NOs. 732 and 734, respectively), respectively.

[0061] The heavy chain domain of Omi12 originates from the IGHV1-58 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi12 but may not contain the light chain of Omi12. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 735, 736, and 737, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi12 (i.e., SEQ ID NO. 732). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 732.

[0062] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi12 but may not contain the heavy chain of Omi12. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 738, 739, and 740, respectively. The antibody may contain or comprise a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi12 (i.e., SEQ ID NO. 734). The antibody may contain or comprise a light chain variable domain containing SEQ ID NO. 734.

[0063] In one embodiment, the antibody in Table 3 may be Omi02. Omi02 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 685, 686, and 687, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 688, 689, and 690, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 682). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 684). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi02 (i.e., SEQ ID NOs. 682 and 684, respectively), respectively.

[0064] The heavy chain domain of Omi02 originates from the IGHV1-69 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi02 but may not contain the light chain of Omi02. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 685, 686, and 687, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi02 (i.e., SEQ ID NO. 682). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 682.

[0065] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi02 but may not contain the heavy chain of Omi02. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 688, 689, and 690, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi02 (i.e., SEQ ID NO. 684). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 684.

[0066] In one embodiment, the antibody in Table 3 may be Omi08. Omi08 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 715, 716, and 717, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 718, 719, and 720, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi08 (i.e., SEQ ID NO: 712). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi08 (i.e., SEQ ID NO: 714). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi08 (i.e., SEQ ID NOs. 712 and 714, respectively), respectively.

[0067] In one embodiment, the antibody in Table 3 may be Omi42. Omi42 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 955, 956, and 957, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 958, 959, and 960, respectively. In one embodiment, the antibody of the present invention may contain or include a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 952). In one embodiment, the antibody of the present invention may contain or include a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 954). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi42 (i.e., SEQ ID NOs. 952 and 954, respectively), respectively.

[0068] The heavy chain domain of Omi42 originates from the IGHV3-9 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi42 but may not contain the light chain of Omi42. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 955, 956, and 957, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi42 (i.e., SEQ ID NO. 952). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 952.

[0069] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi42 but may not contain the heavy chain of Omi42. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 958, 959, and 960, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi42 (i.e., SEQ ID NO. 954). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 954.

[0070] In one embodiment, the antibody in Table 3 may be Omi16. Omi16 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 745, 746, and 747, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 748, 749, and 750, respectively. In one embodiment, the antibody of the present invention may contain or include a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi16 (i.e., SEQ ID NO: 742). In one embodiment, the antibody of the present invention may contain or include a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi16 (i.e., SEQ ID NO: 744). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi16 (i.e., SEQ ID NOs. 742 and 744, respectively), respectively.

[0071] The heavy chain domain of Omi16 originates from the IGHV3-66 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi16 but may not contain the light chain of Omi16. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 745, 746, and 747, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi16 (i.e., SEQ ID NO. 742). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 742.

[0072] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi16 but may not contain the heavy chain of Omi16. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 748, 749, and 750, respectively. The antibody may contain or comprise a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi16 (i.e., SEQ ID NO. 744). The antibody may contain or comprise a light chain variable domain containing SEQ ID NO. 744.

[0073] In one embodiment, the antibody in Table 3 may be Omi18. Omi18 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 765, 766, and 767, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 768, 769, and 770, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi18 (i.e., SEQ ID NO: 762). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi18 (i.e., SEQ ID NO: 764). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi18 (i.e., SEQ ID NOs. 762 and 764, respectively), respectively.

[0074] The heavy chain domain of Omi18 originates from the IGHV3-53 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi18 but may not contain the light chain of Omi18. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 765, 766, and 767, respectively. The antibodies may contain or consist of a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi18 (i.e., SEQ ID NO. 762).

[0075] The antibody may contain or comprise a heavy chain variable domain containing SEQ ID NO: 762.

[0076] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi18 but may not contain the heavy chain of Omi18. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 768, 769, and 770, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi18 (i.e., SEQ ID NO. 764). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 764.

[0077] In one embodiment, the antibodies in Table 3 may be Omi20. Omi20 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 775, 776, and 777, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 778, 779, and 780, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 772). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 774). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi20 (i.e., SEQ ID NOs. 772 and 774, respectively), respectively.

[0078] The heavy chain domain of Omi20 originates from the IGHV3-66 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi20 but may not contain the light chain of Omi20. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 775, 776, and 777, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi20 (i.e., SEQ ID NO. 772). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 772.

[0079] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi20 but may not contain the heavy chain of Omi20. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 778, 779, and 780, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi20 (i.e., SEQ ID NO. 774). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 774.

[0080] In one embodiment, the antibodies in Table 3 may be Omi23. Omi23 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 785, 786, and 787, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 788, 789, and 790, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 782). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 784). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi23 (i.e., SEQ ID NOs. 782 and 784, respectively), respectively.

[0081] The heavy chain domain of Omi23 originates from the IGHV4-31 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi23 but may not contain the light chain of Omi23. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 785, 786, and 787, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi23 (i.e., SEQ ID NO. 782). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 782.

[0082] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi23 but may not contain the heavy chain of Omi23. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 788, 789, and 790, respectively. The antibody may contain or comprise a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi23 (i.e., SEQ ID NO. 784). The antibody may contain or comprise a light chain variable domain containing SEQ ID NO. 784.

[0083] In one embodiment, the antibodies in Table 3 may be Omi28. Omi28 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 835, 836, and 837, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 838, 839, and 840, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 832). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 834). In one embodiment, the antibody of the present invention may contain or comprise amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with the heavy chain variable domain and light chain variable domain of antibody Omi28 (i.e., SEQ ID NOs. 832 and 834, respectively). The heavy chain domain of Omi28 originates from the IGHV3-66 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibody of the present invention may contain the heavy chain of Omi28 but may not contain the light chain of Omi28. For example, the antibody may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 835, 836, and 837, respectively.The antibody may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the heavy chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 832). The antibody may contain or comprise a heavy chain variable domain comprising SEQ ID NO: 832.

[0084] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi28 but may not contain the heavy chain of Omi28. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 838, 839, and 840, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi28 (i.e., SEQ ID NO. 834). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 834.

[0085] In one embodiment, the antibody in Table 3 may be Omi39. Omi39 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 935, 936, and 937, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 938, 939, and 940, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi39 (i.e., SEQ ID NO: 932). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi39 (i.e., SEQ ID NO: 934). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi39 (i.e., SEQ ID NOs. 932 and 934, respectively), respectively.

[0086] In one embodiment, the antibody in Table 3 may be Omi17. Omi17 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 755, 756, and 757, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 758, 759, and 760, respectively. In one embodiment, the antibody of the present invention may contain or include a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi17 (i.e., SEQ ID NO: 752). In one embodiment, the antibody of the present invention may contain or include a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi17 (i.e., SEQ ID NO: 754). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi17 (i.e., SEQ ID NOs. 752 and 754, respectively), respectively.

[0087] The heavy chain domain of Omi17 originates from the IGHV3-66 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi17 but may not contain the light chain of Omi17. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 755, 756, and 757, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi17 (i.e., SEQ ID NO. 752). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 752.

[0088] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi17 but may not contain the heavy chain of Omi17. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 758, 759, and 760, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi17 (i.e., SEQ ID NO. 754). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 754.

[0089] In one embodiment, the antibodies in Table 3 may be Omi29. Omi29 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 845, 846, and 847, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 848, 849, and 850, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 842). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 844). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi29 (i.e., SEQ ID NOs. 842 and 844, respectively), respectively.

[0090] The heavy chain domain of Omi29 originates from the IGHV3-53 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi29 but may not contain the light chain of Omi29. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 845, 846, and 847, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi29 (i.e., SEQ ID NO. 842). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 842.

[0091] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi29 but may not contain the heavy chain of Omi29. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 848, 849, and 850, respectively. The antibody may contain or comprise a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi29 (i.e., SEQ ID NO. 844). The antibody may contain or comprise a light chain variable domain containing SEQ ID NO. 844.

[0092] In one embodiment, the antibody in Table 3 may be Omi36. Omi36 has been found to neutralize the pseudoviral constructs of live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 915, 916, and 917, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 918, 919, and 920, respectively. In one embodiment, the antibody of the present invention may contain or include a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 912). In one embodiment, the antibody of the present invention may contain or include a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 914).

[0093] In one embodiment, the antibody of the present invention may contain or comprise an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with the heavy chain variable domain and light chain variable domain of antibody Omi36 (i.e., SEQ ID NOs. 912 and 914, respectively). The heavy chain domain of Omi36 originates from the IGHV3-66 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibody of the present invention may contain the heavy chain of Omi36 but may not contain the light chain of Omi36. For example, the antibody may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 915, 916, and 917, respectively. The antibody may contain or comprise a heavy chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the heavy chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 912). The antibody may contain or comprise a heavy chain variable domain comprising SEQ ID NO: 912.

[0094] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi36 but may not contain the heavy chain of Omi36. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 918, 919, and 920, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi36 (i.e., SEQ ID NO. 914). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 914.

[0095] In one embodiment, the antibody in Table 3 may be Omi38. Omi38 has been found to neutralize the pseudoviral constructs of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta, and Omicron, as well as Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, and Omicron BA.3 (see Tables 13 and 14, and Figure 2). In one embodiment, the antibodies of the present invention may include CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 925, 926, and 927, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 928, 929, and 930, respectively. In one embodiment, the antibody of the present invention may contain or comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi38 (i.e., SEQ ID NO: 922). In one embodiment, the antibody of the present invention may contain or comprise a light chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Omi38 (i.e., SEQ ID NO: 924). In one embodiment, the antibody of the present invention may contain, or comprise, amino acid sequences having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with respect to the heavy chain variable domain and light chain variable domain of antibody Omi38 (i.e., SEQ ID NOs. 922 and 924, respectively), respectively.

[0096] The heavy chain domain of Omi38 originates from the IGHV1-69 v region, and the inventors have previously demonstrated that swapping of the heavy and light chains between antibodies derived from the same v region results in antibodies particularly useful for the present invention (as further described below). Therefore, the antibodies of the present invention may contain the heavy chain of Omi38 but may not contain the light chain of Omi38. For example, the antibodies may contain CDRH1, CDRH2, and CDRH3 having the amino acid sequences specified in SEQ ID NOs. 925, 926, and 927, respectively. The antibodies may contain or comprise a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the heavy chain variable domain of antibody Omi38 (i.e., SEQ ID NO. 922). The antibodies may contain or comprise a heavy chain variable domain containing SEQ ID NO. 922.

[0097] Alternatively, in one embodiment of the present invention, the antibody may contain the light chain of Omi38 but may not contain the heavy chain of Omi38. For example, the antibody may contain CDRL1, CDRL2, and CDRL3 having the amino acid sequences specified in SEQ ID NOs. 928, 929, and 930, respectively. The antibody may contain or comprise a light chain variable domain having an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with respect to the light chain variable domain of antibody Om38 (i.e., SEQ ID NO. 924). The antibody may contain or comprise a light chain variable domain comprising SEQ ID NO. 924.

[0098] The mixed chain antibody of the present invention The antibody of the present invention may comprise a light chain variable domain containing CDRL1, CDRL2, and CDRL3 derived from a first antibody in Table 1, 2, or 3, and a heavy chain variable domain containing CDRH1, CDRH2, and CDRH3 derived from a second antibody in Table 1, 2, or 3, provided that the first and second antibodies are different. Such an antibody is referred to herein as a mixed-chain antibody.

[0099] Tables 4 to 12 show examples of mixed-chain antibodies useful for the present invention. Table 4 shows examples of mixed-chain antibodies produced from antibodies in Tables 1 to 3 derived from the same germline heavy chain IGHV3-53. Table 5 shows examples of mixed-chain antibodies produced from antibodies in Tables 1 to 3 derived from the same germline heavy chains IGHV3-53 and IGHV3-66. Table 6 shows examples of mixed-chain antibodies produced from antibodies in Tables 1 to 3 derived from the same germline heavy chain IGHV1-58. Table 7 shows examples of mixed-chain antibodies produced from antibodies in Tables 2 and 3 derived from the same germline heavy chain IGHV1-69. Table 8 shows examples of mixed-chain antibodies produced from antibodies in Tables 1 to 3 derived from the same germline heavy chain IGHV3-30. Table 9 shows examples of mixed-chain antibodies produced from antibodies in Tables 2 and 3 derived from the same germline heavy chain IGHV3-33. Table 10 shows examples of mixed-chain antibodies produced from antibodies in Tables 1-3 derived from the same germline heavy chain IGHV1-18. Table 11 shows examples of mixed-chain antibodies produced from antibodies in Tables 1 and 3 derived from the same germline heavy chain IGHV3-9. Table 12 shows examples of mixed-chain antibodies produced from antibodies in Tables 2 and 3 derived from the same germline heavy chain IGHV4-31. Examples of mixed-chain antibodies derived from the same germline heavy chain IGHV1-69 include Omi02H / beta-49L and Omi38H / Omi24L.

[0100] Therefore, in one embodiment, the antibody of the present invention includes a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3 derived from a first antibody in Table 1, 2, or 3, and a light chain variable domain comprising CDRL1, CDRL2, and CDRL3 derived from a second antibody in Table 1, 2, or 3, provided that the first and second antibodies are different. The antibody may include a heavy chain variable domain amino acid sequence having at least 80% sequence identity with the heavy chain variable domain derived from the first antibody in Table 1, 2, or 3, and a light chain variable domain amino acid sequence having at least 80% sequence identity with the light chain variable domain derived from the second antibody in Table 1, 2, or 3, provided that the first and second antibodies are different. For example, an antibody may contain a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the heavy chain variable domain of an antibody in Table 1, 2, or 3, and a light chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the light chain variable domain of an antibody in Table 1, 2, or 3, provided that the first and second antibodies are different.

[0101] The first antibody may be found in Table 3, and the second antibody may also be found in Table 3.

[0102] The first antibody may be found in Table 3, and the second antibody may be found in Table 1. The first antibody may be found in Table 3, and the second antibody may be found in Table 2. The first antibody may be found in Table 1, and the second antibody may be found in Table 3. The first antibody may be found in Table 2, and the second antibody may be found in Table 3. The first antibody may be found in Table 1, and the second antibody may be found in Table 2. The first antibody may be found in Table 2, and the second antibody may be found in Table 1. The first antibody may be found in Table 2, and the second antibody may be found in Table 2.

[0103] In one embodiment, at least one of the first and second antibodies is an antibody from Table 3.

[0104] In one embodiment, the first and second antibodies are not both found in Table 1. In one embodiment, the first and second antibodies are not both found in Table 2. In one embodiment, the first and second antibodies are not both selected from antibodies in Table 1 or 2.

[0105] In one embodiment, at least one of the heavy chain variable domains and the light chain variable domain is from Table 3.

[0106] The antibodies in Table 3 may be selected from the following group: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39, and Omi42. The antibodies in Table 3 may be selected from the following group: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36, and Omi38. For example, the antibodies in Table 3 may be selected from the group consisting of Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, and Omi08. The antibodies in Table 3 may be selected from the group consisting of Omi03, Omi12, Omi02, Omi39, and Omi42. The antibodies listed in Table 3 may be selected from the group consisting of Omi03 and Omi12.

[0107] In one embodiment, both the first and second antibodies are selected from the group consisting of: Omi03, Omi18, Omi29, Beta-27, antibody 150, antibody 158, antibody 175, antibody 222, and antibody 269. The heavy chain variable domains of these antibodies are derived from IGHV3-53. The resulting mixed chain antibodies are listed in Table 4. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% sequence identity to the corresponding variable domain of any one of the mixed chain antibodies listed in Table 4.

[0108] Mixed-chain antibodies can be generated using antibodies derived from IGHV3-53 with antibodies derived from IGHV3-66 (e.g., antibodies 40 and 398 in Table 1) (e.g., Dejnirattisai, Wanwisa, et al. "The antigenic anatomy of SARS-CoV-2 receptor binding domain." Cell 184(8)(2021):2183-2200; Supasa, Piyada, et al. "Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera." Cell 184(8)(2021):2201-2211; Zhou, Daming, et al. "Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera." Cell 184(9)(2021):2348-2361;Dejnirattisai,Wanwisa,et al.“Antibody evasion by the P.1 strain of SARS-CoV-2.”Cell 184(11)(2021):2939-2954;Liu,Chang,et al.“Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.”Cell 184(16)(2021):4220-4236). Therefore, in one embodiment, both the first and second antibodies are selected from the group consisting of: Omi03, Omi18, Omi29, Omi16, Omi17, Omi20, Omi27, Omi36, Beta-27, Antibody 150, Antibody 158, Antibody 175, Antibody 222, Antibody 269, Antibody 40, and Antibody 398. The heavy chain variable domains of these antibodies are derived from IGHV3-53 and IGHV3-66. The resulting mixed-chain antibodies are listed in Table 5.Therefore, the antibody of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies listed in Table 5.

[0109] In one embodiment, both the first and second antibodies are selected from the group consisting of: Omi12, beta-47, beta-25, antibody 55, antibody 165, antibody 253, and antibody 318. The heavy chain variable domains of these antibodies are derived from IGHV 1-58. The resulting mixed chain antibodies are listed in Table 6. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies listed in Table 6.

[0110] In one embodiment, both the first and second antibodies are selected from the group consisting of: beta-49, beta-50, Omi02, Omi24, Omi30, Omi31, Omi34, and Omi38. The heavy chain variable domains of these antibodies are derived from IGHV 1-69. The resulting mixed chain antibodies are listed in Table 7. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies listed in Table 7.

[0111] In one embodiment, both the first and second antibodies are selected from the group consisting of: Beta 22, Beta 29, Antibody 159, and Omi 09. The heavy chain variable domains of these antibodies are derived from IGHV 3-30. The resulting mixed chain antibodies are described in Table 8. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies described in Table 8.

[0112] In one embodiment, both the first and second antibodies are selected from the group consisting of: beta-20, beta-43, Omi32, and Omi33. The heavy chain variable domains of these antibodies are derived from IGHV 3-33. The resulting mixed chain antibodies are listed in Table 9. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies listed in Table 9. The CDRL1-3 of Omi32 and Omi33 are identical, meaning that they are substantially already exemplary mixed chain antibodies of the present invention.

[0113] In one embodiment, both the first and second antibodies are selected from the group consisting of: antibody 278, beta 44, omi 26, and omi 41. The heavy chain variable domains of these antibodies are derived from IGHV 1-18. The resulting mixed chain antibodies are described in Table 10. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies described in Table 10.

[0114] In one embodiment, both the first and second antibodies are selected from the group consisting of: antibody 58, Omi25, Omi35, and Omi42. The heavy chain variable domains of these antibodies are derived from IGHV 3-9. The resulting mixed chain antibodies are described in Table 11. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies described in Table 11.

[0115] In one embodiment, both the first and second antibodies are selected from the group consisting of: Beta 56 and Omi 23. The heavy chain variable domains of these antibodies are derived from IGHV 4-31. The resulting mixed chain antibodies are described in Table 12. Thus, the antibodies of the present invention may contain all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each containing or consisting of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with respect to the corresponding variable domain of any one of the mixed chain antibodies described in Table 12.

[0116] The constant region domain of the antibody molecule of the present invention, if present, may be selected in consideration of candidate functions of the antibody molecule, particularly effector functions that may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG, or IgM domain. Typically, the constant region is of human origin. In particular, a human IgG (i.e., IgG1, IgG2, IgG3, or IgG4) constant region domain may be used. Typically, the constant region is the human IgG1 constant region.

[0117] The specific antibody of the present invention The present invention also provides antibodies that are full-length antibodies of any one of the antibodies in Tables 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In other words, the antibodies of the present invention comprise a heavy chain variable domain and a light chain variable domain, each consisting of a heavy chain variable domain and a light chain variable domain of any one of the antibodies in Tables 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and an IgG (e.g., IgG1) constant region.

[0118] For example, the antibodies of the present invention may be full-length Omi02, Omi03, Omi12, Omi18, Omi28, Omi39, or Omi42 antibodies. The antibodies of the present invention may also be full-length Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36, or Omi38 antibodies. All of these antibodies are very potent neutralizing mAbs that have been shown to neutralize SARS-CoV-2 omicron variants with an IC50 of 0.1 μg / ml or less. These antibodies also have the ability to neutralize at least SARS-CoV-2 Victoria, Alpha, Beta, Gamma, and Delta strains with an IC50 of 0.1 μg / ml or less.

[0119] The antibody may be derived from germline heavy chain IGHV1-58 and contains proline at position 53 of the heavy chain variable region (according to absolute numbering). For example, the antibody may contain an amino acid sequence or a heavy chain variable domain consisting of such a sequence, having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the heavy chain variable domain of Omi-12 (SEQ ID NO: 731), Beta-47 (SEQ ID NO: 591), Beta-25 (SEQ ID NO: 461), Antibody 55 (SEQ ID NO: 62), Antibody 165 (SEQ ID NO: 182), Antibody 253 (SEQ ID NO: 262), or Antibody 318 (SEQ ID NO: 332), provided that the amino acid at position 53 of the heavy chain variable region is proline (according to absolute numbering). For example, the antibodies may include the heavy chain and light chain variable regions of Beta-47 (SEQ ID NOs. 591 and 592, respectively), Beta-25 (SEQ ID NOs. 461 and 462, respectively), Antibody 55 (SEQ ID NOs. 62 and 61, respectively), Antibody 165 (SEQ ID NOs. 182 and 181, respectively), Antibody 253 (SEQ ID NOs. 262 and 261, respectively), or Antibody 318 (SEQ ID NOs. 332 and 331, respectively), except that they have a V53P mutation in the heavy chain variable region. The inventors have found that such antibodies are particularly effective against the Omicron strain (see, for example, Example 5).

[0120] Position 53 in the heavy chain variable region of antibodies Omi-12, beta-47, beta-25, antibody 55, antibody 165, antibody 253, and antibody 318, derived from IGHV1-58, corresponds to position 58 in IMGT numbering.

[0121] Accordingly, the present invention also provides an antibody derived from the germline heavy chain IGHV1-58 that can bind to the spike protein of the coronavirus SARS-CoV-2, wherein the amino acid at position 58 of the heavy chain variable region according to IMGT numbering is proline or substituted with proline.

[0122] The antibody may contain a heavy chain variable domain that has ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the heavy chain variable domain of the antibody derived from germline heavy chain IGHV1-58, provided that the amino acid at position 58 according to IMGT numbering is either proline or substituted with proline.

[0123] Antibodies derived from germline heavy chain IGHV1-58 may be AZD8895, Omi-12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253, or Antibody 318. The amino acid sequences of the heavy chain variable domains of Omi-12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253, or Antibody 318 are described herein (see, for example, Tables 1-3). The amino acid sequence of the heavy chain variable domain of antibody AZD8895 is provided in SEQ ID NO: 963.

[0124] The IGHV1-58 germline V gene sequence encodes the following amino acid sequence: MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAA (SEQ ID NO: 961). Accordingly, the present invention also provides an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising a heavy chain variable domain containing an amino acid sequence having ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with SEQ ID NO: 961, provided that the amino acid at position 58 according to IMGT numbering is proline or substituted with proline.

[0125] The antibody may contain a heavy chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to SEQ ID NOs. 731, 591, 461, 62, 182, 262, or 332, provided that the amino acid at position 58 according to IMGT numbering is proline or substituted with proline. The antibody may contain a heavy chain variable domain containing an amino acid sequence having SEQ ID NOs. 591, 461, 62, 182, 262, or 332, where valine at position 58 according to IMGT numbering is substituted with proline.

[0126] The antibody may contain a heavy chain variable domain with an amino acid sequence having sequence number 963, where the isoleucine at position 58 according to IMGT numbering is substituted with proline.

[0127] In some embodiments, antibodies derived from germline heavy chain IGHV1-58 include a light chain variable domain derived from IGLV kappa 3-20. The antibody may include a light chain variable domain containing an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with the light chain variable domain of the germline IGLV kappa 3-20 antibody. The germline IGLV kappa 3-20V sequence may encode the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO: 967). Therefore, antibodies derived from germline heavy chain IGHV1-58 may contain light chain variable domains that have an amino acid sequence having ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity with SEQ ID NO: 967.

[0128] The present invention also provides an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising a heavy chain variable domain containing an amino acid sequence that is a modified version of SEQ ID NO: 961, provided that the amino acid at position 58 according to IMGT numbering is proline or substituted with proline. The modified version of SEQ ID NO: 961 may include the modifications described herein, e.g., substitutions, deletions and / or additions. For example, modifications may include substitutions and / or deletions of ≤50, ≤45, ≤40, ≤35, ≤30, ≤25, ≤20, ≤15, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2 or 1 amino acid from SEQ ID NO: 961. Modifications may include substitutions and / or deletions of ≤4, ≤3, ≤2 or 1 amino acid from SEQ ID NO: 961.

[0129] The antibody may contain a heavy chain variable domain containing an amino acid sequence that is a modified version of SEQ ID NOs. 731, 591, 461, 62, 182, 262, 332, or 963, including ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or 1 modification, provided that the amino acid at position 58 according to IMGT numbering is proline or substituted with proline. The modified versions of SEQ ID NOs. 731, 591, 461, 62, 182, 262, 332, or 963 may include the modifications described herein, such as substitutions, deletions, and / or additions.

[0130] Antibodies may contain an IgG (e.g., IgG1) constant region.

[0131] The present invention also provides a method for preparing such antibodies. For example, this method may involve modifying an antibody derived from germline heavy chain IGHV1-58, which can bind to the spike protein of the coronavirus SARS-CoV-2, by substituting the amino acid at position 58 of the heavy chain variable region (according to IMGT numbering) with proline. Antibodies derived from germline heavy chain IGHV1-58 may be AZD8895, Omi-12, beta-47, beta-25, antibody 55, antibody 165, antibody 253, or antibody 318. The amino acid sequences of the heavy chain variable domains of each of these antibodies are described herein (see, for example, Tables 1-3 and SEQ ID NO: 963). The present invention also provides antibodies obtained or acquired by this method.

[0132] Characteristics of the antibody of the present invention The antibodies of the present invention may be or may include modifications thereof to the amino acid sequences of the antibodies in Tables 1-12, while maintaining the activity and / or function of the antibody. Modifications may include substitutions, deletions, and / or additions. For example, modifications may include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions derived from the amino acid sequences of the antibodies in Tables 1-12. For example, modifications may include amino acids substituted with alternative amino acids having similar properties. Some properties of the 20 key amino acids that can be used to select appropriate substituents are as follows:

[0133] [Table 1]

[0134] Modifications may include derivatized amino acids, such as labeled or unnatural amino acids, provided that the antibody function is not significantly negatively affected.

[0135] The antibody modifications of the present invention described above can be prepared by modification during or after the synthesis of the antibody, or when the antibody is in a recombinant form using known techniques such as site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0136] The antibodies of the present invention can be modified (for example, as described above) to enhance their potency or to adapt them to a new SARS-CoV-2 variant. The modification may be an amino acid substitution that adapts the antibody to a substitution within the viral variant. For example, known binding modes of the antibody to the spike protein (e.g., by crystallography or modeling) can be used to identify amino acids of the antibody that interact with the substitution in the viral variant. This information can then be used to identify possible antibody substitutions that would compensate for changes in epitope characteristics. For example, a substitution of a hydrophobic amino acid in the spike protein with a negatively altered amino acid can be compensated by substituting an antibody-derived amino acid that interacts with the amino acid in the spike protein with a positively charged amino acid. This disclosure includes methods for identifying antibody residues that can be substituted, for example, by determining the structure of the antibody-antigen complex described herein.

[0137] The antibodies of the present invention may contain one or more modifications that enhance cross-strain neutralization properties. For example, E484 of the spike protein, a key residue that mediates interaction with ACE2, is mutated in several SARS-CoV-2 strains (e.g., the Victoria strain contains E484, while the P.1 and B.1.351 strains contain E484K), resulting in different neutralization effects of the antibody. Therefore, antibodies that bind to E484 may be modified to compensate for the change in E484 of the spike protein. For example, E484 is mutated from a positively charged amino acid to a negatively charged amino acid in the B.1.351 or P.1 strains of SAR-CoV-2 compared to the original strain. Amino acid residues of antibodies that bind to or near E484 may be mutated to compensate for the change in charge. Examples of such amino acid residues may be G104 and / or K108 in SEQ ID NO: 102 of antibody 88 or R52 in SEQ ID NO: 372 of antibody 384.

[0138] The antibody of the present invention may be an isolated antibody. An isolated antibody is an antibody that is substantially free from other antibodies with different antigen specificities.

[0139] As used herein, the term “antibody” may refer to the entire antibody (i.e., including the elements of two heavy chains and two light chains linked together by disulfide bonds) and its antigen-binding fragment. An antibody typically comprises an immunoactive portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds to (and immunely reacts with) an antigen. “Specifically binds” or “immunely reacts with” means that the antibody reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and at least one heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The VH and VL regions can be further subdivided into hypervariability regions called complementarity determination regions (CDRs), into which more conserved regions called framework regions (FRs) are inserted.

[0140] Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single-chain, Fab, Fab' and F(ab')2 fragments, scFv, and Fab expression libraries.

[0141] The antibodies of the present invention may be monoclonal antibodies. Monoclonal antibodies (mAbs) of the present invention can be produced by various techniques, including conventional monoclonal antibody methodologies, such as those disclosed in “Monoclonal Antibodies: a manual of techniques” (Zola H, 1987, CRC Press) and “Monoclonal Hybridoma Antibodies: techniques and applications” (Hurrell JGR, 1982, CRC Press). The antibodies of the present invention may be multispecific, for example, bispecific. The bispecific antibodies of the present invention bind to two different epitopes. The epitopes may be in the same protein (e.g., two epitopes in the SARS-CoV-2 spike protein) or in different proteins (e.g., one epitope in the SARS-CoV-2 spike protein and one epitope in another protein (e.g., a coding protein)).

[0142] In one embodiment, the bispecific antibody of the present invention may bind to two separate epitopes on the spike protein of SARS-CoV-2. The bispecific antibody may bind to the NTD and RBD of the spike protein. The bispecific antibody may bind to two different epitopes within the RBD of the spike protein.

[0143] One or more (e.g., two) antibodies of the present invention can be coupled to form a multispecific (e.g., bispecific) antibody. Methods for preparing multispecific antibodies, such as bispecific antibodies, are well known in the art.

[0144] Antibodies may be selected from the group consisting of single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen-binding regions (Fab), recombinant antibodies, monoclonal antibodies, fusion proteins containing the antigen-binding domain or aptamer of a native antibody, single-domain antibodies (sdAb), also known as VHH antibodies, nanobodies (single-domain antibodies derived from camelids), single-domain antibody fragments derived from shark IgNAR called VNAR, diabodies, triabodies, antikalin, aptamers (DNA or RNA), and their active portions or fragments.

[0145] The constant region domain of the antibody molecule of the present invention, if present, may be selected in consideration of candidate functions of the antibody molecule, particularly effector functions that may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG, or IgM domain. Typically, the constant region is of human origin. In particular, a human IgG (i.e., IgG1, IgG2, IgG3, or IgG4) constant region domain may be used. Typically, the constant region is the human IgG1 constant region.

[0146] The light chain constant region can be lambda or kappa.

[0147] The antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies typically comprise at least two distinct variable domains, each of which can bind to a different antigen or to a different epitope on the same antigen.

[0148] The antibodies of the present invention may be chimeric antibodies, CDR-grafted antibodies, nanobodies, human antibodies, or humanized antibodies. Typically, the antibodies are human antibodies. Fully human antibodies are antibodies in which the variable regions and constant regions (if present) of the heavy and light chains are all of human origin, or are substantially identical to human sequences but not necessarily derived from the same antibody.

[0149] The antibody of the present invention may be a full-length antibody.

[0150] The antibody of the present invention may be an antigen-binding fragment. The antigen-binding fragment of the present invention binds to the same epitope of the parent antibody, i.e., the antibody from which the antigen-binding fragment originates. The antigen-binding fragment of the present invention typically retains a portion of the parent antibody that interacts with the epitope. The antigen-binding fragment typically contains complementarity-determining regions (CDRs) that interact with the antigen, e.g., 1, 2, 3, 4, 5, or 6 CDRs. In some embodiments, the antigen-binding fragment further includes a structural scaffold surrounding the CDRs of the parent antibody, e.g., variable region domains of the heavy and / or light chains. Typically, the antigen-binding fragment retains the same or similar binding affinity to the antigen as the parent antibody.

[0151] Antigen-binding fragments do not necessarily have the same sequence as the parent antibody. In one embodiment, the antigen-binding fragment may have ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with each CDR of the parent antibody. In one embodiment, the antigen-binding fragment may have ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, and 100% sequence identity with each variable region domain of the parent antibody. Typically, non-identical amino acids in the variable region are not present in the CDR.

[0152] The antigen-binding fragments of the antibodies of the present invention retain the ability to selectively bind to antigens. Examples of antigen-binding fragments of antibodies include single-chain antibodies (i.e., full-length heavy chains and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), and scFv.

[0153] The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Methods for preparing and manufacturing these antibody fragments are well known in the art (see, for example, Verma R et al., 1998, J.Immunol.Methods, 216, 165-181).

[0154] Methods for screening antibodies of the present invention that do not share 100% amino acid sequence identity with one of the antibodies disclosed herein and that have desired specificity, affinity, and functional activity include methods described herein, such as enzyme-linked immunosorbent assays, ViaCore, focus reduction neutralization assays (FRNT), and other techniques known in the art.

[0155] In terms of function, the antibody of the present invention can neutralize at least one biological activity of SAR-CoV-2 (neutralizing antibody), in particular, it can neutralize viral transmissibility.

[0156] Neutralization can also be determined using the IC50 or IC90 value. For example, antibodies have IC50 values ​​of ≤0.1 μg / ml, ≤0.05 μg / ml, ≤0.01 μg / ml, ≤0.005 μg / ml, or ≤0.002 μg / ml. In some cases, the antibodies of the present invention may have an IC50 value of 0.0001 μg / ml to 0.1 μg / ml, sometimes 0.0001 μg / ml to 0.05 μg / ml or 0.0001 μg / ml to 0.001 μg / ml.

[0157] For example, the IC50 values ​​of some of the antibodies in Tables 1-12 are provided in Tables 13-16.

[0158] The ability of an antibody to neutralize viral transmissibility can be measured using appropriate assays, particularly cell-based neutralization assays, as shown in the examples. For example, neutralization ability can be measured by a focus reduction neutralization assay (FRNT), in which the reduction in the number of virus-infected cells (e.g., human cells) in the presence of the antibody (e.g., after 2 hours at 37°C) is compared to a negative control to which the antibody was not added.

[0159] The antibodies of the present invention can block the interaction of the SAR-CoV-2 spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) of target cells, for example, by direct blocking or by disrupting the pre-fusion higher-order structure of the spike protein.

[0160] Blocking of the interaction between spike and ACE2 can be whole or partial. For example, the antibodies of the present invention can reduce spike-ACE2 formation by ≧50%, ≧60%, ≧70%, ≧80%, ≧90%, ≧95%, ≧99% or 100%. Blocking of spike-ACE2 formation can be measured by any suitable means known in the art, such as ELISA.

[0161] Most antibodies showing neutralization also showed blocking of the interaction between the spike protein and ACE2. Furthermore, some non-neutralizing antibodies are excellent ACE2 blockers.

[0162] From the perspective of binding kinetics, the antibodies of the present invention can have an affinity constant (KD) value of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.5 nM, ≦0.4 nM, ≦0.3 nM, ≦0.2 nM or ≦0.1 nM for the spike protein of SARS-CoV-2.

[0163] The KD value can be measured by any suitable means known in the art, such as ELISA or surface plasmon resonance (Biacore) at 25°C.

[0164] Binding affinity (KD) can be quantified by determining the dissociation constant (Kd) and the association constant (Ka) for the antibody and its target. For example, the antibody can have an association constant (Ka) of ≧10000 M -1 s -1 ≧50000 M -1 s -1 ≧100000 M -1 s -1 ≧200000 M -1 s -1 or ≧500000 M -1 s -1 and / or a dissociation constant (Kd) of ≦0.001 s -1 ≦0.0005 s -1 ≦0.004 s -1 ≦0.003 s -1 ≦0.002 s -1 or ≦0.0001 s-1 It is possible.

[0165] The antibodies of the present invention can preferably provide in vivo protection in animals infected with coronaviruses (e.g., SARS-CoV-2). For example, administration of the antibodies of the present invention to animals infected with coronaviruses (e.g., SARS-CoV-2) may result in survival rates of ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, or 100%. Survival rates can be determined using conventional methods.

[0166] The antibodies of the present invention may have any combination of one or more of the above-mentioned properties.

[0167] The antibodies of the present invention may bind to the same epitope as any of the antibodies described herein, or may compete for binding to the SARS-CoV-2 spike protein (i.e., particularly with respect to antibodies having the heavy and light chain variable regions described above). Methods for identifying antibodies that bind to the same epitope or that cross-compete with each other are used in the examples and are discussed further below.

[0168] Fc area The antibody of the present invention may or may not contain an Fc domain.

[0169] The antibodies of the present invention may be modified within the Fc region to improve their stability. Such modifications are known in the art. Modifications can improve the stability of the antibody during storage. The in vivo half-life of the antibody can be improved by modification of the Fc region. For example, the introduction of a cysteine ​​residue into the Fc region enables the formation of interchain disulfide bonds within this region. Homodimerated antibodies thus produced may have improved internal migration ability and / or increased complement-mediated cell death and / or antibody-dependent cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)).

[0170] Alternatively, antibodies can be designed that possess a dual Fc region, thereby enhancing complement lysis and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0171] For example, the antibody of the present invention may be modified to promote the interaction of the Fc domain with FcRn. The Fc domain may be modified to improve antibody stability by influencing the Fc-FcRn interaction at low pH, for example, within endosomes. The M252Y / S254T / T256E(YTE) mutation may be used to improve the half-life of an IgG1 antibody.

[0172] Antibodies can be modified to influence their interaction with other receptors such as FcγRI, FcγRIIA, FcγRIIB, FcγRIII, and FcαR. Such modifications can be used to affect the effector function of the antibody.

[0173] In one embodiment, the antibody of the present invention comprises a modified Fc domain as described herein below. In another preferred embodiment, the antibody of the present invention comprises an Fc domain, but the sequence of the Fc domain is modified to modify one or more Fc effector functions.

[0174] In one embodiment, the antibody of the present invention includes a "silenced" Fc region. For example, in one embodiment, the antibody of the present invention does not exhibit effector function or function associated with a standard Fc region. The Fc region of the antibody of the present invention does not bind to one or more Fc receptors.

[0175] In one embodiment, the antibody of the present invention does not contain a CH2 domain. In one embodiment, the antibody of the present invention does not contain a CH3 domain. In one embodiment, the antibody of the present invention contains an additional CH2 domain and / or CH3 domain.

[0176] In one embodiment, the antibody of the present invention does not bind to the Fc receptor. In another embodiment, the antibody of the present invention does not bind to complement. In an alternative embodiment, the antibody of the present invention does not bind to FcγR but binds to complement.

[0177] In one embodiment, the antibody of the present invention may generally include modifications that alter the serum half-life of the antibody. Therefore, in another embodiment, the antibody of the present invention has Fc region modifications that alter the half-life of the antibody. Such modifications and modifications that alter Fc function may exist. In a preferred embodiment, the antibody of the present invention has modifications that alter the serum half-life of the antibody.

[0178] In one embodiment, the antibody of the present invention may include human constant-state domains, such as IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic applications where antibody effector function is required, human IgG constant-state domains, especially IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, IgG2 and IgG4 isotypes may be used.

[0179] In one embodiment, the antibody heavy chain includes a CH1 domain, and the antibody light chain includes a CL domain (kappa or lambda). In another embodiment, the antibody heavy chain includes a CH1 domain, a CH2 domain, and a CH3 domain, and the antibody light chain includes a CL domain (kappa or lambda).

[0180] The four human IgG isotypes bind to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa), inhibitory FcγRIIb receptor, and the first component of complement (C1q) with different affinities, resulting in very different effector functions (Bruhns P. et al., 2009. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood. 113(16):3716-25). See also Jeffrey B. Stavenhagen, et al. Cancer Research 2007 Sep 15;67(18):8882-90. In one embodiment, the antibody of the present invention does not bind to Fc receptors. In another embodiment of the present invention, the antibody binds to one or more types of Fc receptors.

[0181] In one embodiment, the Fc region used is mutated (particularly the mutations described herein). In one embodiment, the Fc mutation is selected from the group including mutations that eliminate or enhance the binding of the Fc region to the Fc receptor, mutations that increase or eliminate effector function, mutations that prolong or shorten the half-life of the antibody, and combinations thereof. In one embodiment referring to the impact of modification, the Fc mutation may be demonstrated by comparison with an equal antibody lacking the modification.

[0182] Several antibodies that selectively bind to FcRn at pH 6.0 rather than pH 7.4 exhibit longer half-lives in various animal models. Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (Hinton PR. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6) and M252Y / S254T / T256E+H433K / N434F (Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8), have been shown to increase binding affinity to FcRn and extend the half-life of IgG1 in vivo. Therefore, modifications that alter the serum half-life may exist in M252 / S254 / T256+H44 / N434, and in particular, M252Y / S254T / T256E+H433K / N434F may exist. In one embodiment, it is desirable to extend the half-life. In another embodiment, it may actually be desirable to shorten the serum half-life of the antibody, and therefore, modifications that shorten the serum half-life may exist.

[0183] Numerous mutations have been formed within the CH2 domain of human IgG1, and their effects on ADCC and CDC have been studied in vitro (Idusogie EE et al., 2001. Engineered antibodies with increased activity to recruit complement. Immunol. 166(4):2571-5). Visibly, alanine substitution at position 333 has been reported to increase both ADCC and CDC. Therefore, in one embodiment, modifications at position 333, particularly those altering the ability to recruit complement, may exist. Lazar et al. described a triple mutant (S239D / I332E / A330L) with higher affinity for FcγRIIIa and lower affinity for FcγRIIb, resulting in enhanced ADCC (Lazar GA et al., 2006). Therefore, modifications of S239 / I332 / A330, particularly those that alter affinity for the Fc receptor, especially S239D / I332E / A330L, may exist (Engineered antibody Fc variants with enhanced effector function. PNAS 103(11):4005-4010). Using the same mutation, antibodies with increased ADCC were generated (Ryan MC. et al., 2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer Ther., 6:3009-3018). Richards et al. studied the FcγRIIa / FcγRIIb ratio that mediates enhanced phagocytosis of target cells by slightly different triple mutants (S239D / I332E / G236A) with improved FcγRIIIa affinity and macrophages (Richards JO et al 2008. Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 7(8):2517-27).Therefore, in one embodiment, the S239D / I332E / G236A modification may exist.

[0184] In another embodiment, the antibody of the present invention may have a modified hinge region and / or a CH1 region. Alternatively, the isotype used may be selected to have a specific hinge region.

[0185] Major public virtual domains The public V region (also referred to herein as the public V gene) is the V region of the germline heavy and light chain regions found in the majority of antibody responses to SARS-CoV-2 observed in populations. In this context, the V region is a specific response to the beta-SARS-CoV-2 variant. That is, many individuals utilize the same v region from the germline v region repertoire when generating an immune response to the SARS-CoV-2 variant.

[0186] As used herein, an antibody "derived" from a specific v region refers to an antibody produced by V(D)J recombination using that germline v region sequence. For example, the germline IGHV3-53 v region sequence can undergo somatic recombination and somatic mutation to reach an antibody that specifically binds to the SARS-CoV-2 spike protein. The nucleotide sequence encoding the antibody is unlikely to contain the same sequence as the IGHV3-53 germline sequence, but the antibody is still derived from this v region. Antibodies of the present invention typically contain no more than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nonsilent mutations in the v region compared to the germline sequence. Antibodies of the present invention typically do not contain 2 to 20 nonsilent mutations in the v region compared to the germline sequence, for example, 5 to 15, 6 to 13, or 7 to 12 nonsilent mutations. Germline v region sequences are well known in the art, and methods for identifying whether a specific region of an antibody originates from a specific germline v region sequence are also well known in the art.

[0187] In one embodiment, the antibody of the present invention is derived from a v region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV1-69, IGHV3-30, IGHV3-33, IGHV1-18, IGHV13-9, or IGHV4-31. The inventors have found that the potent neutralizing antibodies identified herein contain relatively few mutations within the CDRs of these v regions. Therefore, in one embodiment, the antibody of the present invention is encoded by a v region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV1-69, IGHV3-30, IGHV3-33, IGHV1-18, IGHV13-9, or IGHV4-31, and has 2 to 20 non-silent nucleotide mutations or 5 to 15 non-silent mutations, for example, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 non-silent mutations compared to a naturally occurring germline sequence. A silent mutation as defined herein is a change in a nucleotide sequence that does not result in a change in the amino acid sequence encoded by the nucleotide sequence. Therefore, a non-silent mutation is a mutation that results in a change in the amino acid sequence encoded by the nucleotide sequence.

[0188] The inventors have surprisingly discovered that the light chain variable regions of two antibodies having the same heavy chain v region can be exchanged to produce a mixed-chain antibody containing the heavy chain variable region of the first antibody and the light chain variable region of the second antibody. For example, the two antibodies may both contain heavy chain variable regions derived from IGHV3-53. Preferably, both antibodies also contain light chain variable regions derived from the same light chain v region, but this is not essential, because, for example, the light chain of antibody 222 can match any heavy chain variable region derived from IGHV3-53 to result in a potent neutralizing antibody. As described above, the two antibodies may contain heavy chain variable regions derived from IGHV3-53 and / or IGHV3-66.

[0189] In one embodiment, the antibody of the present invention is an antibody derived from a major public v region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV4-39, IGHV3-30, IGHV5-51, IGHV1-02, or IGHV3-33 (for example, for IGHV3-53 antibodies Omi03, Omi18, Omi29, beta-27, antibody 150, antibody 158, antibody 175, antibody 222 and antibody 269; for IGHV3-66 antibodies Omi16, Omi17, Omi20, Omi27, Omi36, antibody 40 and antibody 398; for IGHV1-58 antibodies Omi12, beta-47, beta-25, antibody This includes CDRs of the heavy chain variable domains of antibodies 55, 165, 253, 1-69 (antibodies Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34, and Omi38), 1-30 (antibodies Beta-22, Beta-29, 159, and Omi09), 3-33 (antibodies Beta-20, Beta-43, Omi32, and Omi33), 1-18 (antibodies 278, Beta-44, Omi26, and Omi41), 3-9 (antibodies 58, Omi25, Omi35, and Omi42), or 4-31 (antibodies Beta-56 and Omi23). The sequence numbers corresponding to each of these antibody CDRs are shown in Tables 1, 2, and 3.

[0190] In one embodiment, the antibody of the present invention is an antibody derived from a major public v region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV4-39, IGHV3-30, IGHV5-51, IGHV1-02, or IGHV3-33 (for example, for IGHV3-53 antibodies Omi03, Omi18, Omi29, beta-27, antibody 150, antibody 158, antibody 175, antibody 222 and antibody 269; for IGHV3-66 antibodies Omi16, Omi17, Omi20, Omi27, Omi36, antibody 40 and antibody 398; for IGHV1-58 antibodies Omi12, beta-47, beta-25, The antibodies 55, 165, 253, and IGHV1-69 contain the heavy chain variable domains of antibodies beta-49, beta-50, Omi02, Omi24, Omi30, Omi31, Omi34, and Omi38; IGHV3-30 contains the heavy chain variable domains of antibodies beta-22, beta-29, 159, and Omi09; IGHV3-33 contains the heavy chain variable domains of antibodies beta-20, beta-43, Omi32, and Omi33; IGHV1-18 contains the heavy chain variable domains of antibodies beta-278, beta-44, Omi26, and Omi41; IGHV3-9 contains the heavy chain variable domains of antibodies beta-58, Omi25, Omi35, and Omi42; or IGHV4-31 contains the heavy chain variable domains of antibodies beta-56 and Omi23. The sequence numbers corresponding to each of these antibodies' CDRs are shown in Tables 1, 2, and 3.

[0191] In one embodiment, the present invention provides a method for generating an antibody that specifically binds to the spike protein of SARS-CoV-2 (e.g., alpha, beta, gamma, delta, and / or omicron lineage SARS-CoV-2 strains), comprising: identifying two or more antibodies derived from the same light chain and / or heavy chain v region; and generating a mixed chain antibody comprising the heavy chain of a first antibody and the light chain of a second antibody by substituting the light chain of a first antibody with the light chain of a second antibody. In one embodiment, the method further comprises determining the affinity of the mixed chain antibody to SARS-CoV-2 and / or its neutralization. The method may further comprise comparing the affinity of the mixed chain antibody with that of the first and / or second antibody. The method may further comprise selecting a mixed chain antibody that has the same or greater affinity than the first and / or second antibody. In some embodiments, the heavy chain v region is IGHV 1-58 and / or the light chain v region is IGLV kappa 3-20.

[0192] In another embodiment, the present invention provides an antibody that specifically binds to an omicron variant of SARS-CoV-2, the antibody having a v region derived from IGHV1-69. Surprisingly, it has been found that the antibody response to infection by the omicron variant of SARS-CoV-2 is biased toward antibodies having a heavy chain variable region derived from IGHV1-69. In one embodiment, when the antibody heavy chain is derived from IGHV1-69, the antibody of the present invention includes CDRH1, CDRH2, and CDRH3 from beta-49, beta-50, Omi02, Omi24, Omi30, Omi31, Omi34, and Omi38.

[0193] Antibody conjugate The present invention also relates to immunoconjugates containing antibodies conjugated to cytotoxic agents, such as toxins (e.g., enzymatically active toxins or fragments thereof of bacterial, fungal, plant, or animal origin) or immunoconjugates containing radioisotopes (i.e., radioconjugates). Conjugates of antibodies and cytotoxic agents can be formed using various bifunctional protein coupling agents known in the art.

[0194] The antibodies of the present invention can be conjugated to molecules that regulate or alter the serum half-life. For example, the antibodies of the present invention can bind to albumin to regulate the serum half-life. In one embodiment, the antibodies of the present invention also include an albumin-specific binding region. In another embodiment, the antibodies of the present invention may include a peptide linker, which is an albumin-binding peptide. Examples of albumin-binding peptides are given in International Publication No. 2015 / 197772 and International Publication No. 2007 / 106120 (both incorporated in their entirety by reference).

[0195] Polynucleotides, vectors, and host cells The present invention also provides one or more isolated polynucleotides (e.g., DNA) encoding the antibody of the present invention. In one embodiment, the polynucleotide sequence may be aggregated on multiple polynucleotides, but can collectively encode the antibody of the present invention. For example, the polynucleotide may encode the heavy chain variable region and / or light chain variable region of the antibody of the present invention. The polynucleotide may encode the full-length heavy chain and / or light chain of the antibody of the present invention. Typically, one polynucleotide encodes the heavy chain and / or light chain, respectively.

[0196] The polynucleotides encoding the antibodies of the present invention can be obtained by methods well known to those skilled in the art. For example, the DNA sequences encoding some or all of the antibody heavy and light chains can be synthesized from the corresponding amino acid sequences, if necessary. General methods for constructing vectors, translocation methods, and culture methods are well known to those skilled in the art. In this regard, see “Current Protocols in Molecular Biology”, 1999, FMAusubel(ed), Wiley Interscience, New York and Maniatis Manual produced by Cold Spring Harbor Publishing. The polynucleotides of the present invention may be provided in the form of expression cassettes that enable in vivo expression of the antibodies of the present invention by including a regulatory sequence operably linked to the inserted sequence. Thus, the present invention also provides one or more expression cassettes encoding one or more polynucleotides encoding the antibodies of the present invention. These expression cassettes are typically then provided in a vector (e.g., a plasmid or recombinant viral vector). Thus, in one embodiment, the present invention provides a vector encoding the antibodies of the present invention. In another embodiment, the present invention provides a vector encoding the antibodies of the present invention collectively. The vector may be a cloning vector or an expression vector. Suitable vectors may be any vector that can contain a sufficient amount of genetic information and enable the expression of the polypeptide of the present invention. The polynucleotide, expression cassette, or vector of the present invention is introduced into a host cell, for example, by translocation. Therefore, the present invention also provides a host cell containing one or more polynucleotides, expression cassettes, or vectors of the present invention. The polynucleotide, expression cassette, or vector of the present invention can be introduced transiently or permanently into a host cell to enable the expression of antibodies from one or more polynucleotides, expression cassettes, or vectors. Examples of such host cells include transient or preferably stable higher eukaryotic cell lines, such as mammalian or insect cells; lower eukaryotic cells, such as yeast or prokaryotic cells; and bacterial cells, for example.Specific examples of cells include mammalian HEK293 cells, such as HEK293F, HEK293T, HEK293S, or HEK Expi293F, CHO, HeLa, NS0, and COS cells, or any other cell lines used herein, such as those used in the examples. Preferably, the selected cell lines are not only stable but also capable of mature glycosylation.

[0197] The present invention also provides an antibody production process comprising culturing host cells containing one or more vectors of the present invention under conditions suitable for the expression of antibodies from one or more polynucleotides of the present invention, and isolating the antibodies from the culture.

[0198] antibody combinations The inventors have found that certain antibodies in Table 3, as well as specific combinations of antibodies in Tables 3, 2, and 1, are particularly effective when used in combination to minimize loss of activity due to SARS-CoV-2 variants, maximize therapeutic effect, and / or increase diagnostic accuracy. Useful combinations include antibodies that do not cross-compete with each other and / or do not bind to non-overlapping epitopes.

[0199] Therefore, the present invention provides a combination of antibodies, each antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, and at least one antibody comprises at least three CDRs of any one of the 28 antibodies in Table 3.

[0200] The antibody combinations of the present invention may be useful as therapeutic cocktails. Therefore, the present invention also provides pharmaceutical compositions containing the antibody combinations of the present invention, as will be further described below.

[0201] The antibody combinations of the present invention may be useful for diagnosis. Therefore, the present invention also provides diagnostic kits comprising the antibody combinations of the present invention. Also provided herein are methods for diagnosing diseases or complications associated with coronavirus infection in a subject, as will be further described below. A fully cross-neutralizing antibody, e.g., Omi03, may be used as a reference to confirm the presence and / or amount of a variant of SARS-CoV-2 of concern (VoC) in a sample. A limited number of antibodies that bind to VoC may be used to confirm the presence and / or amount of VoC in a sample. For example, if Omi03 shows binding to the sample but Omi24 does not show binding of SARS-CoV-2 to the sample, the spike protein may be the spike protein of the delta VoC. This can be determined by any method known to those skilled in the art, e.g., via an immunoassay, e.g., ELISA or immunochromatographic assay. Binding reduction can be determined by comparison and / or normalization to a reference and / or comparison with positive / negative control samples or data.

[0202] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising the antibodies of the present invention. The composition may comprise a combination of the antibodies of the present invention (e.g., two, three, or four). The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.

[0203] The compositions described herein may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects. Examples of such salts include acid addition salts and base addition salts.

[0204] A pharmaceutically acceptable and suitable carrier includes an aqueous carrier or a diluent. Examples of suitable aqueous carriers include water, buffer water, and physiological saline.

[0205] Other suitable pharmaceutically acceptable carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it will be desirable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition.

[0206] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0207] The pharmaceutical composition of the present invention may include additional therapeutic agents, such as antiviral agents. These antiviral agents may bind to the coronavirus and inhibit its viral activity. Alternatively, antiviral agents may not directly bind to the coronavirus but may still affect its viral activity / infectivity. The antiviral agent may be a further anti-coronavirus antibody, which binds somewhere on SARS-CoV-2 other than the spike protein. Examples of antiviral agents useful in the present invention include remdesivir, lopinavir, ritonavir, APN01, and Favilavir.

[0208] Additional therapeutic agents may include anti-inflammatory agents, such as corticosteroids (e.g., dexamethasone) or nonsteroidal anti-inflammatory drugs (e.g., tocilizumab).

[0209] Additional therapeutic agents may include anti-coronavirus vaccines. Pharmaceutical compositions may be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally. Within the scope of the present invention are also kits comprising the antibody or other composition of the present invention and instructions for use. The kit may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents considered herein.

[0210] Method and Use of the Invention The present invention further relates to the use of antibodies, antibody combinations, and pharmaceutical compositions described herein in, for example, methods of treating or diagnosing the body of a human or animal by therapeutic means. The treatment method may be therapeutic or prophylactic.

[0211] For example, the present invention relates to a method for treating coronavirus (e.g., SARS-CoV-2) infection or related diseases or complications, such as COVID-19. This method may include administering a therapeutically effective amount of the antibody, antibody combination, or pharmaceutical composition of the present invention. The method may further include identifying the presence of coronavirus, such as SARS-CoV-2, or fragments thereof, in a sample derived from a subject. The present invention also relates to antibodies, antibody combinations, or pharmaceutical compositions according to the present invention for use in a method for treating coronavirus (e.g., SARS-CoV-2) infection or related diseases or complications, such as COVID-19.

[0212] The present invention also relates to a method for formulating a composition for treating coronavirus (e.g., SARS-CoV-2) infection, and related diseases or complications, such as COVID-19, the method comprising preparing the composition by mixing an antibody, combination of antibodies, or pharmaceutical composition according to the present invention with an acceptable carrier.

[0213] The present invention also relates to the use of antibodies, combinations of antibodies, or pharmaceutical compositions according to the present invention for treating coronavirus (e.g., SARS-CoV-2) infection or related diseases or complications, such as COVID-19.

[0214] The present invention also relates to the use of antibodies, combinations of antibodies, or pharmaceutical compositions according to the present invention for the manufacture of agents for treating or preventing coronavirus (e.g., SARS-CoV-2) infection or related diseases or complications, such as COVID-19.

[0215] This invention also relates to the prevention, treatment, or diagnosis of coronavirus infection caused by any SARS-CoV-2 strain. Coronavirus infection can be caused by any SARS-CoV-2 strain.

[0216] The SARS-CoV-2 strain may be the earliest identified Wuhan strain (hCoV-19 / Wuhan / WIV04 / 2019(WIV04); GISAID commission no. EPI_ISL_402124) and its variants. For example, the SARS-CoV-2 strain may be a member of lineages A, A.1, A.2, A.3, A.5, B, B.1, B.1.1, B.2, B.3, B.4, B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), delta, kappa and / or lambda. SARS-CoV-2 strains may be members of lineages A.23.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.258, B.1.526.2, B.1.616, B.1.617.1 (kappa), B.1.617.2 (delta), C36.3, C.37 (lambda), P.1 (gamma), B.1.1.529 (omicron), omicron BA.1, omicron BA.1.1, omicron BA.2 and / or omicron BA.3.

[0217] Compared to hCoV-19 / Wuhan / WIV04 / 2019(WIV04) (GISAID request no. EPI_ISL_402124), SARS-CoV-2 strains may contain one or more mutations, for example, within the spike protein. In other words, a SARS-CoV-2 strain may be a modified hCoV-19 / Wuhan / WIV04 / 2019(WIV04) strain that contains one or more modifications within the spike protein.

[0218] This mutation could be a mutation (e.g., a substitution) observed in the Omicron strain of SARS-CoV-2.

[0219] The antibodies Omi02, Omi03, Omi12, Omi18, Omi28, Omi39, and Omi42 are particularly effective in neutralizing the Omicron SARS-CoV-2 strain. Therefore, the present invention may relate to these antibodies for use in the treatment, prevention, management, or diagnosis of coronavirus infections caused by the SARS-CoV-2 strain.

[0220] The methods and uses of the present invention may include inhibiting a disease state (e.g., COVID-19), for example, slowing its progression; and / or mitigating a disease state (e.g., COVID-19), for example, causing remission of the disease state until a desired endpoint is reached.

[0221] The methods and uses of the present invention may include improving or reducing the severity, duration, or frequency of symptoms of a disease condition (e.g., COVID-19) (e.g., reducing pain or discomfort), and such improvement may or may not directly affect the disease. Symptoms or complications may include fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, respiratory tract infection, cytokine storm, acute respiratory distress syndrome (ARDS), sepsis, and / or organ failure (e.g., heart, kidney, liver, GI, lung).

[0222] The methods and uses of the present invention can lead to a reduction in the viral load of coronaviruses (e.g., SARS-CoV-2) by, for example, ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or 100% compared to pretreatment. Methods for determining viral load are well known in the art, and include, for example, infection assays.

[0223] The methods and uses of the present invention may include preventing the occurrence of coronavirus infection in subjects (e.g., humans) who have a predisposition to contract complications associated with coronavirus infection.

[0224] The present invention also relates, for example, to identifying subjects who have coronavirus infection caused by SARS-CoV-2. For example, the methods and uses of the present invention may include identifying the presence of coronavirus (e.g., SARS-CoV-2) or its proteins or fragments in a sample. Detection may be performed in vitro or in vivo. In certain embodiments, the present invention relates to population screening.

[0225] The present invention relates to the identification of any SARS-CoV-2 strain as described herein. The present invention may also relate to a method for identifying an escape mutant of SARS-CoV-2, comprising contacting a sample with a combination of antibodies of the present invention and determining whether each antibody binds to the virus. The term “escape mutant” refers to a variant of SARS-CoV-2 containing a non-silent mutation that may affect the effectiveness of existing treatments for SARS-CoV-2 infection. Typically, the non-silent mutation is on an epitope recognized by prior art antibodies and / or antibodies described herein that specifically bind to an epitope of SARS-CoV-2, for example, on the spike protein of SARS-CoV-2. If an antibody does not bind to the target, this may indicate that the target contains a mutation that may alter the effectiveness of existing treatments for SARS-CoV-2.

[0226] The method and use of the present invention may include contacting a sample with the antibody or combination of antibodies of the present invention and detecting the presence or absence of an antibody-antigen complex, the presence of which indicates that the subject is infected with SARS-CoV-2.

[0227] Methods for determining the presence of antibody-antigen complexes are known in the art. For example, in vitro detection techniques include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vivo techniques involve introducing labeled antianalyte protein antibodies into a target. For example, the antibody can be labeled with a radioactive marker whose presence and location within the target can be detected by standard imaging techniques. Depending on the assay used, the detection technique may provide a qualitative or quantitative readout.

[0228] Typically, the present invention relates to methods and uses for human subjects that require them. However, non-human animals, such as rats, rabbits, sheep, pigs, cattle, cats, or dogs, are also intended. Subjects, such as healthcare workers or people who have come into contact with infected individuals, may be at risk of exposure to coronavirus infection. Subjects may have visited or be scheduled to visit countries known or suspected to have had a coronavirus outbreak. Subjects, such as immunocompromised individuals, such as those receiving immunosuppressive therapy or those suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), may also be at greater risk. Subjects may be asymptomatic or pre-symptomatic.

[0229] The target group may be in the early, middle, or late phases of the disease.

[0230] The subjects may be hospitalized or present in the community at the time of the initial manifestation, and / or may be hospitalized thereafter.

[0231] The subject may be male or female.

[0232] In certain embodiments, the subjects are typically male. The subjects may not be infected with a coronavirus, such as SARS-CoV-2. The subjects may have a predisposition to develop more severe symptoms or complications associated with coronavirus infection. The methods or uses of the present invention may include the step of determining whether a patient is in a condition at risk of developing more severe symptoms or complications associated with coronavirus.

[0233] In embodiments of the present invention relating to prevention or treatment, the subject may or may not be diagnosed with infection with a coronavirus, such as SARS-CoV-2.

[0234] The present invention relates to the analysis of samples derived from a subject. The sample may be tissues, cells, and biological fluids isolated from the subject and the tissues, cells, and fluids present within the subject. The sample may be blood and fractions or components of blood, including serum, plasma, or lymph. Typically, the sample is derived from a throat swab, nasal swab, or saliva.

[0235] Antibody-antigen complex detection assays may be performed in situ, in which case the sample is a (fixed and / or frozen) tissue section obtained from a biopsy or excision of the subject.

[0236] Antibody drugs In embodiments of the present invention in which compositions and combinations are administered, they may be administered subcutaneously, intravenously, intradermally, orally, intranasally, intramuscularly, or intracranially. Typically, antibody pharmaceutical compositions and combinations are administered intravenously or subcutaneously.

[0237] The antibody dose may vary depending on the age and size of the subject, as well as the disease, administration conditions, and route. Antibodies may be administered in doses ranging from approximately 0.1 mg / kg body weight to approximately 100 mg / kg body weight, for example, from approximately 5 mg / kg to approximately 10 mg / kg. Antibodies may also be administered in doses of approximately 50 mg / kg body weight, 10 mg / kg, or approximately 5 mg / kg.

[0238] The combinations of the present invention may be administered, for example, at doses of approximately 5 mg / kg to approximately 10 mg / kg per antibody, or at doses of approximately 10 mg / kg or approximately 5 mg / kg per antibody. Alternatively, the combinations may be administered at a total dose of approximately 5 mg / kg (for example, a combination of three antibodies resulting in a dose of 1.67 mg / kg for each antibody).

[0239] The antibody or combination of antibodies of the present invention may be administered in a multi-dose regimen. For example, the initial dose may be followed by a second or subsequent dose. The second and subsequent doses may be divided at appropriate intervals.

[0240] As described above, the antibodies of the present invention are typically used in a single pharmaceutical composition / combination (co-formulated). However, the present invention generally also includes the combined use of the antibodies of the present invention in separate formulations / combinations. The present invention also includes the combined use of antibodies with additional therapeutic agents, as described above.

[0241] The combined administration of two or more agents and / or antibodies can be achieved in several different ways. In one embodiment, all components may be administered together in a single composition. In another embodiment, each component may be administered separately from part of the combination therapy.

[0242] For example, the antibody of the present invention may be administered before, after, or in parallel with another antibody or its conjugated fragment of the present invention. Particularly useful combinations are described above, for example.

[0243] For example, the antibody of the present invention may be administered before, after, or concurrently with an antiviral or anti-inflammatory agent.

[0244] In embodiments relating to the present invention for detecting the presence of coronaviruses, such as SARS-CoV-2, or their proteins or fragments, in a sample, the antibody contains a detectable label. For example, methods of attaching a label to an antibody are known in the art, such as direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody. Alternatively, the antibody may be indirectly labeled, for example, by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end labeling of a biotin-tipped DNA probe that can be detected by fluorescently labeled streptavidin.

[0245] The detection may further include (i) an agent known to be useful in detecting the presence of coronaviruses, e.g., SARS-CoV-2 or its proteins or fragments, e.g., an antibody against another epitope of the spike protein or an antibody against another coronavirus protein, e.g., an anti-nucleocapsid antibody; and / or (ii) an agent known to be unable to detect the presence of coronaviruses, e.g., SARS-CoV-2 or its fragments, i.e., an agent that provides a negative control.

[0246] In certain embodiments, the antibody is modified to increase its stability. Appropriate modifications are described previously.

[0247] The present invention also encompasses kits for detecting the presence of coronaviruses, such as SARS-CoV-2, in a sample. For example, a kit may include: a labeled antibody or combination of labeled antibodies of the present invention; means for determining the amount of coronavirus, such as SARS-CoV-2, in a sample; and means for comparing the amount of coronavirus, such as SARS-CoV-2, in a sample with a standard. The labeled antibody or combination of labeled antibodies can be packaged in a suitable container. The kit may further include instructions for using the kit for detecting coronaviruses, such as SARS-CoV-2, in a sample. The kit may further include other agents known to be useful in detecting the presence of coronaviruses, as previously described.

[0248] For example, the antibodies or combinations of antibodies of the present invention are used in lateral flow tests. Typically, a lateral flow test kit is a handheld device comprising a series of capillary beds, e.g., pieces of porous paper, and absorbent pads based on microstructured polymers or sintered polymers. The test involves running a liquid sample along the surface of a pad having reactive molecules to produce a visible positive or negative result. The test may further include using other agents known to be useful in detecting the presence of coronaviruses, e.g., SARS-CoV-2 or fragments thereof, as previously described, such as anti-nucleocapsid antibodies.

[0249] others It should be understood that the various uses of the antibody combinations or pharmaceutical compositions disclosed in this invention can be adapted to specific needs in the art. It should also be understood that the terminology used herein is solely for the purpose of describing specific embodiments of the invention and is not intended to limit them. In addition, the singular forms “a,” “an,” and “it” used herein and in the appended claims include multiple references unless explicitly indicated otherwise. For example, a reference to “antibody” includes two or more “antibodies.”

[0250] Furthermore, where "≧x" is referred to herein, it means that x is greater than or equal to x. Where "≦x" is referred to herein, it means that x is less than or equal to x.

[0251] For the purposes of the present invention, to determine the percent identity of two sequences (e.g., two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for optimal alignment with the second sequence). Subsequently, the nucleotides or amino acid residues at each position are compared. If a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotide or amino acid is identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100). Typically, sequence comparisons are performed over the entire length of the reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be used as the reference sequence. To evaluate whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), one of ordinary skill in the art would perform an alignment over the entire length of SEQ ID NO: 3 to determine what number of positions within the test sequence are identical to the positions in SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, gaps or missing positions should be considered non-identical positions. One of ordinary skill in the art is aware of the various computer programs available for determining homology or identity between two sequences. For example, comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm incorporated into the GAP program within the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ) using either the Blosum 62 matrix or the PAM250 matrix along with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0252] The CDRs of the heavy chain variable domain (CDRH) and the CDRs of the light chain variable domain (CDRL) are located at residues 27-38 (CDR1), residues 56-65 (CDR2), and residues 105-117 (CDR3) of each chain according to the IMGT numbering system (http: / / www.imgt.org; Lefranc MP, 1997, J, Immunol. Today, 18, 509). Unless otherwise indicated, this numbering system is used herein.

[0253] Regardless of the above or below, all publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0254] The following examples illustrate the present invention.

Example

[0255] Example 1. Generation of Antibodies Specific for Early Pandemic SARS-CoV-2 and Beta SARS-CoV-2 Strains The antibodies in Table 1 relate to a set of mAbs generated against early pandemic strains of SARS-CoV-2. The antibodies in Table 2 are related to a set of mAbs generated against the beta strain of SARS-CoV-2.

[0256] Further details of these antibodies can be found in International Patent Applications PCT / GB2022 / 050306 and PCT / GB2022 / 050307. Detailed information on the production and characterization of these antibodies can be found in the following publications: Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184.8(2021):2183-2200. Supasa, Piyada, et al. “Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.” Cell 184.8(2021):2201-2211. Liu,Chang,et al. “The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants.”Cell hostµbe(2021). Zhou, Daming, et al. “Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.” Cell 184.9(2021):2348-2361. Dejnirattisai, Wanwisa, et al. “Antibody evasion by the P.1 strain of SARS-CoV-2.” Cell 184.11(2021):2939-2954. Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16(2021):4220-4236. Dejnirattisai, Wanwisa, et al. “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.”Cell(2022).

[0257] Example 2. Generation of antibodies specific to the Omicron strain of SARS-CoV-2. Omicron BA.2 system Omicron BA.2 was first reported in South Africa on November 17, 2021, around the same time as the first reported case of Omicron BA.1. BA.2 is increasing compared to BA.1 in several countries, including Denmark, India, and the UK, and currently accounts for the majority of Omicron infections in Denmark. While there is accumulating evidence that BA.2 is more contagious than BA.1, there is no evidence that it increases the severity of the disease.

[0258] BA.2 is related to BA.1 and shares 21 amino acid substitutions spread throughout S, but there are some differences. BA.1 has 6 additional amino acid deletions, 3 insertions, and 9 substitutions compared to BA.2, while BA.2 has 3 additional deletions and 7 substitutions compared to BA.1. In RBD, BA.1 contains the unique mutations S371L, G446S, and G496S, and some isolates contain R346K (BA.1.1), while BA.2 contains S371F, T376A, D405N, and R408S. All of these residues may have different effects on antibody binding and may modulate neutralization. These are BA.1 G446S, G496S, BA.2 D405N, and R408S, particularly at the edges of the ACE2 binding footprint. For BA.1.1, the R346K mutation is near the N343 glycan and may modulate potent antibody binding to this region. BA.3 appears to be a fusion of two molecules, not containing the intrinsic mutations associated with BA.1 and BA.2, resembling BA.1 at the N-terminus and switching to a BA.2-like mutation at the C-terminus via the G496S mutation.

[0259] Omicron lines BA.4 and BA.5 In early April 2022, two new Omicron lines were reported from Gauteng Province, South Africa, and named BA.4 and BA.5. The S sequences of BA.4 and BA.5 are identical and closely related to BA.2. The sequence diversity of Omicron S is shown in Figure 9. Compared to BA.2, BA.4 has deletions of residues 69 and 70 and contains two additional substitutions in the RBD: L452R and F486V. Finally, BA.4 lacks the Q493R change seen in BA.1 and BA.2, and reverts to Q493, similar to the Victoria / Wuhan strain.

[0260] The two additional mutations in RBD are of most concern from the perspective of antibody escape: L452R is a chemically radical change and is one of a pair of changes in delta RBD (the other, T478K, has already been found in the Omicron lineage). The mutation F486L was found in the sequence of SARS-CoV-2 isolated from mink in the early stages of the pandemic and is also the site of escape mutations against several mAbs (Gobeil et al., 2021, “Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity”. Science 373, 6555). The F486V change in BA.4 / 5 is also a decrease in the bulk of the hydrophobic side chain, similar to F486L, but more pronounced. Both residues 452 and 486 are located near the edge of the ACE2 interaction surface (Figure 9B), and both, along with reversion to the ancestral sequence Q493 within the ACE2 footprint, have the potential to modulate ACE2 affinity and regulate the neutralizing ability of vaccines or naturally acquired serum. The L452R and F486V mutations are likely to further increase antibody escape, while reversion at 493 may reduce the aforementioned escape from the response to the virus.

[0261] Omicron series BA.2.75 In early May 2022, a new omicron BA.2 sublineage, named BA.2.75, was reported in India. It has since spread to several countries, including the UK, the US, Australia, Germany, and Canada. BA.2.75 contains multiple mutations in the S protein compared to BA.2, including four substitutions within the NTD (W152R, F157L, I210V, G257S) and four substitutions within the RBD: D339H, G446S, N460K, and R493Q (Figure 16). The RBD mutations may act on the major epitopes of neutralizing antibodies and modulate ACE2 binding. D339H represents a further evolution of the G339D mutation found in all previous omicron variants and has been found to inhibit the binding of certain "right-hand" antibodies (e.g., beta-49 and -50) belonging to the IGHV1-69 family. It also corresponds to the binding footprint of certain class 3 antibodies such as S309 / sotolovimab (Dejnirattisai et al., 2022; “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell 185, 467-484 e415). G446S was found in BA.1, BA.1.1, and BA.3, but not in BA.2 or other BA.2 subvariants. It can also inhibit the binding of certain class 3 antibodies that bind to the right shoulder, such as REGN10987 / imdevimab (Dejnirattisai et al., 2022). R493Q reactivation was also seen in BA.4 / 5, which may make the virus more susceptible to neutralization by some class 1 and 2 antibodies that bind to the neck / left shoulder. This reactivation may also increase affinity for ACE2 (see below).

[0262] N460K is a novel mutation not seen in previous VoC or Omicron sublineages, but it was discovered after in vitro (yeast display) evolution in RBD-62, which has extremely high ACE2 affinity (KD = 16-18 pM) (Dejnirattisai et al., 2022; Zahradnik et al., 2021 “SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution”. Nat Microbiol 6, 1188-1198). In fact, N460K resulted in a significant increase in affinity for ACE2, and its effect was second only to N501Y (Zahradnik et al., 2021). Furthermore, in silico analysis predicted that N460K may affect the binding of certain antibodies belonging to the IGHV3-53 family (e.g., Omi-3), and it has been shown to be able to potently neutralize all VoC (Nutalai et al., 2022).

[0263] Neutralization assays showed that delta infection alone does not provide protection (neutralization) against BA.2.75. Mutations in BA.2.75 reduce the neutralizing titer of vaccine serum compared to BA.2. Individual mutations in BA.2.75 can cause a significant decrease in neutralizing titer compared to the complete BA.2.75S sequence, but these are balanced by the R393Q revert mutation, which may have been selected to increase affinity for ACE2 and enhance the transmissibility of BA.2.75. Further evolution of the omicron lineage is inevitable, and there appear to be many trade-offs between antibody escape and ACE2 affinity, which likely leads to successive waves of infection.

[0264] Subtypes of BA.2, BA.4, and BA.5 that appeared Several lineages are rapidly growing from both the BA.2 and BA.5 branches. Most notably, there is a high degree of convergent evolution, particularly at antigen RBD locations such as 346, 444, 452, 460, 486, 490, 493, and 494. These lineages include those from the BA.4 / 5 branch (naturally containing L452R, F486V, and reverted R493Q), e.g., BA.4.6 and BF.7 (R346T), BA.4.7 (R346S), BQ.1 (K444T, N460K), and BQ.1.1 (R346T, K444T, N460K); and the BA.2.75 branch (G339H, G446S, N460K, and reverted R493Q). Examples include those from BA.2.75.2 (R346T and F486S and BA.2.75 mutations), BN.1 (also known as BA.2.75.5.1, which includes R346T, K356T, F490S and BA.2.75 mutations), and BM.1.1.1 (also known as BA.2.75.3.1.1.1, which includes R346T, F486S, F490S and BA.2.75 mutations). Examples of several other second-generation BA.2 variant strains include, for example, BJ.1 (also known as BA.2.10.1.1; G339H, R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (reverted to G446S, F486P, S494P and R493Q), BS.1 (also known as BA.2.3.2.1; R346T, L452R, N460K, G476S and There are also the following: Q493 (reintroduced), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and Q493R reintroduced), and finally, BJ.1×BM.1.1.1 (also known as BA.2.75.3.1.1.1) recombinant, and XBB (which includes R346T, L368I, V445P, G446S, N460K, F486S, F490S and Q493 reintroduced compared to BA.2).

[0265] Outside the RBD, convergent evolution is still present, albeit at a lower degree. Many second-generation BA.2 variant lines contain deletions or mutations in the NTD, often similar to those found in the VoC, such as Δ144 in BJ.1, BS.1, and BA.2.10.4 (previously seen in alpha and BA.1), and NSP12 G671S in BJ.1, XBB, and BA.2.10.4 (previously seen in delta).

[0266] Potent neutralizing antibodies isolated after omicron infection Five volunteers who had recovered from omicron infection, as confirmed by sequencing, were recruited, and samples were collected 10–14 days after symptom onset. All volunteers had received two doses of Pfizer BioNtech vaccine prior to omicron infection. First, neutralization assays were performed against omicron BA.1 and Victoria (an early pandemic SARS-CoV-2 isolate containing only one amino acid substitution at S NTD(S247R) compared to the Wuhan strain sequence used in all current vaccines). In all cases, the focus reduction neutralization 50% titer (FRNT50) against omicron was above 100, but at this early stage, the titer was considerably lower than that against Victoria (Figure 1A).

[0267] B cells from five donors were stained with full-length BA.1 trimers, and single cells were classified by FACS (Figure 1B). After degenerate RT-PCR, the heavy and light chain sequences were assembled into expression vectors using the Gibson reaction, and the products were transfected into 293T cells. The culture supernatant was screened for reactivity to full-length BA.1 or wild-type S (WT Wuhan) along with BA.1 RBD and NTD. A total of 1,122 single cells were classified, and 545 mAbs were recovered.

[0268] All mAbs showed cross-reactivity between WT and BA.1S by ELISA, suggesting that they may have been generated from memory B cells induced by vaccination. In contrast to past panels of monoclonal antibodies, monoclonal antibodies were produced from naive cases infected early in the pandemic (Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184.8 (2021): 2183-2200), and the proportion of Omicron-specific mAbs reacting with the RBD (56%) was found to be higher compared to mAbs from the early pandemic (21%, p<0.0001) (Figure 1C). Furthermore, (Figure 1C). Furthermore, 129 out of 545 isolated mAbs bound to the BA.1 NTD.

[0269] Isolation of Potent Omicron mAbs Neutralization assays were performed on all ELISA-positive mAbs, and those showing the highest activity were selected for further study. The 28 most potent mAbs were selected for full characterization, and all of them showed a BA.1 FRNT50 titer < 100 ng / ml. 27 / 28 bound to the RBD (one Omi-41 bound to the NTD), and none cross-reacted with the SARS-CoV-1 S protein in ELISA.

[0270] Investigation of gene usage (Figure 1D, Table 17) revealed that 9 / 28 mAbs belong to the VH3-53 and related VH3-66 gene families. VH3-53 and VH3-66 have been repeatedly isolated in SARS-CoV-2 infections, and they form a public antibody response and play a role in binding to the neck region of the RBD and blocking ACE2 binding. Previously, many VH3-53 and VH3-66 mAbs were observed to lose activity against VoCs containing the N501Y mutation, but some VH3-53 antibodies (mAb222 and beta-27) were completely resistant to the N501Y changes seen in alpha, beta, and gamma, but their activity against Omicron BA.1 or BA.2 was knocked down.

[0271] Approximately half of the gene families observed in the potent early pandemic antibodies (Table 1) are also presented in the Omicron set (Figure 1C), and perhaps the most striking difference is that VH1-69 is not found in the early antibodies but is present in 6 / 28 (2, 24, 30, 31, 34, and 38) of the potent Omicron set, which we also found in two beta antibodies, beta 49 and 50, which bind to a site adjacent to the N343 glycan. Analysis of the Omicron mAbs shows that the CDR3 sequence is much longer for beta 49 and 50, suggesting a different binding mode. In the beta set of mAbs, the expansion of the public reaction was found to be mediated via VH4-39 (6 / 27 mAb) which binds to the epitope around the 501Y mutation, although most lose activity against BA.1. It is noteworthy that none of the current set of Omicron mAbs are encoded by VH4-39.

[0272] Compared to the initial pandemic antibody set, the inventors found higher levels of somatic mutations in both the heavy and light chains of mAb omicrons compared to the initial pandemic set (average VH and VL levels were 9.00 and 6.00, respectively, compared to 4.55 and 4.25 in the initial pandemic). These results are considered consistent with the evolution of increased omicron affinity due to somatic mutations in vaccine-induced memory B cells.

[0273] Broad-spectrum neutralization of VoC by Omicron mAb Neutralization assays were performed on a panel of 28 potent mAbs against Victoria and all variants of concern (alpha, beta, gamma, delta, and omicron BA.1) (Figures 2A-C, Tables 13-16 and 18). The high probability that all these antibodies originated from vaccine-induced memory B cells is evident from the fact that in almost all cases, the FRNT50 titer against Victoria was at the upper limit of all VoCs tested with each mAb (Figures 2A-C, Tables 13-16 and 18). Five mAbs neutralized BA.1 with FRNT50 titers <10 ng / ml, and mAbs Omi-3, 8, 12, 18, and 24 were the most potent with FRNT50 titers of 9, 8, 4, 6, and 7 ng / ml, and FRNT90 titers of 67, 42, 20, 18, and 35 ng / ml, respectively.

[0274] The data provided in Tables 13, 14, and 16 include some IC50 data obtained using dummy virus constructs. The data in Table 18 consists of IC50 results obtained only from true virus constructs.

[0275] Antibodies 17 / 28 were cross-reactive against all VoCs, and the difference in FRNT50 titers between all viruses was less than 10-fold. Omi-06, 24, 30, 31, 34, and 41 showed reduced or no activity against delta, and 3 / 6 of these belonged to the VH1-69 family and may have an epitope affecting the L452R delta mutation (delta and BA.1 share T478K). Antibodies Omi-09 and 32 performed poorly against beta and gamma and may be sensitive to E484K seen in beta and gamma, but can tolerate the E484A variation in omicron (omicron shares N501Y and K417N with beta, while gamma has N501Y and K417T). Finally, 129 anti-NTD mAbs were isolated, but only one of them, Omi-41, showed an FRNT50 titer of less than 100 ng / ml. Omi-41 showed neutralizing activity against Victoria, Alpha, Beta, and Gamma, but not against Delta. This is thought to be due to the unique spectrum of the NTD changes observed in Delta.

[0276] Neutralization of BA.1 compared to BA.1.1, BA.2, and BA.3 Lentivirus-based reporters were constructed by pseudotyped with the S gene sequences of Victoria, BA.1, BA.1.1, BA.2, and BA.3. Neutralization assays against omicron mAbs are shown in Figure 2B, Tables 14 and 18, and most antibodies showed little difference in neutralization of BA.1, BA.1.1, BA.2, and BA.3. However, there were some notable exceptions. Neutralization of BA.2 was reduced 38-fold, 3-fold, and 158-fold in Omi-8, 29, and 32 compared to BA.1, respectively, while neutralization of BA.1.1 was reduced 40.9-fold, 10.8-fold, 7.8-fold, and 6.6-fold in Omi-6, 24, 34, and 35 compared to BA.1, respectively, and knocked out in Omi-39 and 40. Neutralization of BA.3 by Omi-mAbs was very similar to that observed with BA.2, with the exception of Omi-06 and Omi-36, which showed significantly lower BA.3 neutralizing titers than BA.1 or BA.2. For some reason, the NTD-binding mAb Omi-41 did not neutralize Victoria in the pseudovirus system, but it did neutralize live virus. This was also observed with the early pandemic mAb 159, which showed potent activity against live virus but no activity against pseudovirus.

[0277] Pseudoviral neutralization curves for a panel of mAbs isolated from early pandemic cases and mAbs isolated from beta cases are shown in Figures 4A, B, and Table 15. In most cases, the neutralization titers for BA.1, BA.1.1, and BA.2 are similar, but there are some differences: mAbs 40, 278, and 318 neutralize BA.2 better than BA.1, while 222, beta 22, 29, 54, 55, and 56 neutralize BA.1 better than BA.2, and beta 53, which binds near the N343 glycan, shows reduced neutralization of BA.1.1.

[0278] Neutralization using antibodies developed for clinical use. Finally, neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 strains was tested using mAbs developed for clinical use, and several differences were found (Figure 2C, Tables 16 and 18). Interestingly, the activity of known antibody A (REGN10987) was partially restored in BA.2, but still decreased 308-fold compared to Victoria; the activity of known antibody C (AZD1061) was almost completely restored in BA.2; known antibody D (AZD8895) decreased 5.4-fold in BA.2 compared to BA.1; and both combinations of known antibodies D (AZD8895) and E decreased only 8-fold compared to Victoria. The activity of known antibody K (S309) decreased 6.8-fold in BA.2 compared to BA.1, and finally, the activity of known antibody G (ADG20) was completely lost in BA.2.

[0279] In summary, the neutralization of most omicron monoclonal antibodies is not affected by differences between BA.1, BA.1.1, BA.2, or BA.3 mutations. However, some monoclonal antibodies, particularly known antibodies A (REGN10987) and C (AZD1061), which neutralize BA.2 more readily than BA.1, and known antibody K (S309), which shows reduced neutralization of BA.2, exhibit differences, and sub-system typing before use may be recommended. A structural explanation of the differences between BA.1, BA.1.1, BA.2, and BA.3 neutralization is provided below.

[0280] Neutralization of BA.1, BA.1.1, BA.2, and BA3 with immunoserum To determine whether the difference in transmissibility between BA.1 and BA.2 may be due to differences in neutralization, and whether BA.2 may escape the BA.1 antibody response, neutralization assays were performed using serum from various sources. First, neutralization assays were performed for Victoria, BA.1, BA.1.1, BA.2, and BA.3 using serum collected from vaccinated individuals who received Oxford / AstraZeneca AZD1222 (n=41) or Pfizer / BioNtech BNT162b2 (n=20) vaccines (Figure 3A, B).

[0281] For AZD1222, samples were collected 4 weeks after the second and third doses of the vaccine. After the third dose of AZD1222, there was a small but significant difference in pseudoviral neutralization with decreased titers for BA.2 vs. BA.1 (1.17 times, p=0.0019) and BA.1.1 vs. BA.1 (1.29 times, p=0.0086). For BNT162b2, samples were collected 4 weeks and 6 months after the second dose of the vaccine, before the third dose, and 4 weeks after the third dose. After the third dose of the vaccine, titers for BA.1, BA.2, and BA.1.1 were similar, with no significant differences among them. Next, the neutralization profiles of serum collected from cases infected with Omicron were determined. Early samples (n=12) were collected less than 14 days (median 13 days) after symptom onset, and late samples (n=17) were collected more than 21 days (median 38 days) after symptom onset. All cases received at least two doses of vaccine, and some late-recovery cases received a third dose after omicron infection. Neutralization against Victoria, Alpha, Beta, Gamma, Delta, and Omicron was tested using live virus neutralization assays (Figure 3C). At the early time point, all vaccinated cases showed high titers against Victoria, with a geometric mean FRNT50 close to 1 / 3000, and broad neutralization of VoC with FRNT50 > 1 / 1000 for all viruses except Omicron (FRNT50 = 558). At subsequent time points, titers against Victoria did not change, but titers against VoC and Omicron increased (3-fold, p = 0.0123). Pairwise comparison of early and late-stage samples taken from the same individual revealed a widespread enhancement of the response after omicron infection (Figure 5A).

[0282] The neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 was assayed by pseudoviral neutralization. The neutralizing titer of BA.1 was higher at later time points. However, all serum was obtained from cases of BA.1 infection, and the neutralizing titer of BA.2 versus BA.1 was slightly but significantly decreased (1.7-fold and 1.5-fold at <14 days and >21 days, p=0.0034 and 0.0067), and the titer of BA.1.1 versus BA.1 did not decrease significantly, but at >21 days, the titer of BA.3 versus BA.1 decreased 1.7-fold (p=0.0012) (Figures 3D, 5B).

[0283] In summary, three doses of the vaccine, particularly BNT162b2, induced good neutralizing titers of antibodies against omicron BA.1, BA.1.1, BA.2, and BA.3, with only slight differences in titers for BA.1, BA.1.1, BA.2, and BA.3. This may indicate that the increased transmissibility of BA.2 is not due to increased vaccine escape. Following the breakthrough in omicron infection, individuals who have been previously vaccinated show enhanced broad antibody responses against variants of concern, resulting in a strong response to omicron. Because there are only slight differences in the neutralization of BA.1 and BA.2, it is considered unlikely, at least in the short term, that co-infection with BA.2 will occur in cases exposed to BA.1 and subsequently vaccinated.

[0284] Neutralization of BA.4 compared to BA.1, BA.1.1, BA.2, and BA.3 Neutralization of BA.4 / 5 was evaluated compared to the Omicron sublineages BA.1, BA.1.1, BA.2, BA.3, and the early pandemic Victoria strain. BA.4 / 5 was shown to have a more extreme antibody escape phenotype than BA.1 and BA.2, with neutralizing titers approximately 2–3 times lower in serum from donors who received three doses of vaccine compared to neutralization of BA.1 and BA.2. Furthermore, serum from breakthrough BA.1 infection in vaccinated individuals showed approximately 2–3 times lower neutralizing titers against BA.4 / 5 compared to BA.1 and BA.2. This suggests that currently approved vaccines and mAbs may have low efficacy in preventing transmission of BA.4 / 5. Therefore, new monoclonals and combinations may be needed to fill this gap in order to protect extremely vulnerable individuals and those unable to initiate an adequate vaccine response.

[0285] Neutralization of BA.4 by vaccine serum Panels of pseudotyped lentiviruses expressing the S gene of the Omicron sublineages BA.1, BA.1.1, BA.2, BA.3, and BA.4 / 5 (Di Genova et al., 2020, “Production, titration, neutralization and storage of SARS-CoV-2 lentiviral pseudotypes”. Figshare preprint) were constructed together with Victoria, an early Wuhan-related strain of the pandemic, which was used as a control.

[0286] The neutralization assay was performed using serum obtained 28 days after the third dose of either the Oxford-AstraZeneca vaccine ADZ1222 (n=41) (Flaxman et al., 2021. "Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002)". Lancet 398, 981-990.) or the Pfizer-BioNtech vaccine BNT162b2 (Cele et al., 2021; "Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization".) Nature 602, 654-666) (n=20). (Figure 7A, B). For AZD1222, the neutralizing titer of BA.4 was 2.1 times lower compared to BA.1 (p=0.0001) and 1.8 times lower compared to BA.2 (p=0.0001). For BNT162b2, the neutralizing titers were 3.2 times (p=0.0001) and 3.1 times (p=0.0001) lower compared to BA.1 and BA.2, respectively. Because antibody titers naturally decline, these decreases in titers, especially in the long term, may reduce the effectiveness of the vaccine.

[0287] Breakthrough: Neutralization of BA.4 / 5 with serum from BA.1 infection When the Omicron outbreak began, vaccinated volunteers who had contracted Breakthrough Omicron infection were recruited. Samples were initially collected within 14 days of symptom onset (median 13 days), and later samples were collected 21 days or more after symptom onset (median 38 days) n=16. Pseudovirus neutralization assays were performed on panels of pseudoviruses representing suspected variants and Omicron sublineages (Figure 7C, D).

[0288] Post-vaccination BA.1 infection resulted in a broad neutralization response with high titers against all VoCs, which was enhanced at later time points (Nutalai et al. 2022, “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”. Cell (in press)). Neutralizing titers against BA.4 were significantly lower than those against BA.1 and BA.2, with BA.4 / 5 titers decreasing 1.9-fold (p=0.0001) and 1.5-fold (p=0.0015) at the initial time point compared to BA.1 and BA.2, respectively. At the later time point, BA.4 / 5 titers decreased 3.4-fold (p=0.0001) and 2-fold (p=0.0017) at the later time point compared to BA.1 and BA.2, respectively.

[0289] Therefore, BA.4 / 5 exhibits a certain degree of immune escape from the vaccine / BA.1 response compared to BA.1 and BA.2. Since all of these samples were collected reasonably close to the time of infection, they may become more susceptible to reinfection with BA.4 / 5 after further decline over the following months.

[0290] Escape from BA.4 / 5 monoclonal antibody Susceptibility to L452R: To date, Omi-24, 30, 31, 34, and 41 have been reported to show complete knockout of neutralizing activity against delta, and Omi-06 has shown severe knockdown of activity (Nutalai et al., 2022). BA.1 and BA.2 contain only one of the two delta RBD mutations (T478K), but BA.4 / 5 also contains L452R, so it is expected that all five L452-targeted mAbs will be knocked out in BA.4 / 5. This has indeed been observed (Figure 8A, Table 20). Omi-41 also fails to neutralize, but this is thought to be due to differences in the NTD mutation (Figure 9A).

[0291] To confirm that the observed neutralization effect was directly attributable to the change in RBD interaction, binding analysis of selected antibodies to BA.4 / 5 and BA.2 RBDs by surface plasmon resonance (SPR) was also performed (Figures 10, 15). Omi-31 was selected as a representative of the L452R-sensitive antibody set, and as expected, binding was severely affected (Figure 10A).

[0292] Since detailed information is available regarding the interaction of several omicron-responsive antibodies with RBDs, the BA.4 / 5 RBD mutation was modeled in relation to known structures of omicron Fabs compounded with BA.1 or delta RBDs (Dejnirattisai et al., 2022; “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses”. Cell 185, 467-484 e415; Nutalai et al., 2022), (Figure 11). The Omi-31 complex is shown in Figure 11A, showing that L452 fits snugly into the hydrophobic pocket and cannot accommodate the larger positively charged arginine in BA.4 / 5 and delta.

[0293] L452R enhancement of binding: Omi-32 shows a 77-fold enhancement of BA.4 / 5 neutralization compared to BA.2. Kinetic analysis of Fab binding to RBD suggests that this is mainly achieved by a 5-fold increase in binding rate (Figure 10B, C). This is mainly explained by a favorable interaction in which arginine at 452 forms a salt bridge at residue 99 of the heavy chain (HC)CDR3 (Figure 11B), which may have been facilitated by the removal of a slightly unfavorable charge interaction at residue 493. These electrostatic changes may lead to improved binding rate by electrostatic induction of the incoming antibody.

[0294] Sensitivity to F486V: Extending the theory used to understand delta sensitivity, the remaining antibodies affected by BA.4 / 5 > BA.2 but retaining activity against delta, namely Omi-02, 09, 12, 23, 25, 26, and 29, may be sensitive to the F486V mutation. Binding sensitivity was confirmed by SPR analysis of Omi-12 (Figure 10D, E), showing an almost 1,000-fold decrease in affinity. An example of the structural basis of sensitivity is provided by the Omi-25 complex (Figure 11C), showing that the phenylalanine side chain acts as a binding hotspot, located within a hydrophobic cavity, and forms a favorable ring-stacking interaction with Y106 of HC CDR3.

[0295] Activity of commercially available antibodies against BA.4 and BA.5 A panel of antibodies developed for therapeutic / preventive use was tested against BA.4 / 5 (Figure 8B, Table 21). Many of these antibodies already had significantly reduced or knocked-out activity against BA.1, BA.1.1, or BA.2. For AstraZeneca AZD1061, the activity against BA.4 / 5 was similar to that against BA.2 (less than a twofold decrease), but for AZD8895, the residual activity against BA.2 was knocked out. The activity of the two antibody combination in AZD7442 (Dong et al., 2021, “Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail”. Nature Microbiol. 6, 1233-1244) was 8.1-fold decreased compared to BA.2. The residual activity of REG10987 (Weinreich et al., 2021, “REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19”. N Engl J Med 384, 238-251) was further reduced in BA.4 / 5 compared to BA.2, and similarly, the residual BA.1 neutralizing activity in BA.4 / 5 was knocked out for ADG20 (Yuan et al., 2022, “A broad and potent neutralization epitope in SARS-related coronaviruses”. bioRxiv. https: / / doi.org / 10.1101 / 2022.03.13.484037). Regarding S309 (VIR-7831 / 7832) (Sun and Ho, 2020, “Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic”. Antib Ther 3, 246-256.), its activity against BA.4 / 5 was 1.6 times lower compared to BA.2.

[0296] These effects can be rationalized by referring to how antibodies interact with RBDs, for example, in the case of AZD8895 (IGHV1-58 genotype mAb, Figure 11E), F486 forms a hydrophobic interaction hotspot, which is neutralized by mutations to a much smaller valine side chain. The antibody residues involved in interaction with F486, including Omi-12, 253, and Beta-47, are highly conserved among mAbs of this genotype (Nutalai et al., 2022, “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”.Cell (in press); Dejnirattisai et al., 2021, “The antigenic anatomy of SARS-CoV-2 receptor binding domain”.Cell 184, 2183-2200 e2122; Liu et al., 2021, “The Beta mAb response underscores the antigenic distance to other SARS-CoV-2 variants”.Cell, Host and Microbe 30, 53-68), which explains why the F486V mutation significantly affects the neutralization of these mAbs (Figure 8A, 13).

[0297] Neutralization of BA.2.75 by vaccine serum As described above (Di Genova et al., 2020), a panel of pseudotyped lentiviruses expressing the S gene of the Omicron sublineages BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75 was constructed along with Victoria, an early Wuhan-related strain of the pandemic used as a control. D339H, G446S, N460K, and R493Q were also included as single mutations in the BA.2 background. The neutralization assay was performed using serum obtained 28 days after the third dose of either the Oxford-AstraZeneca vaccine AZD1222 (n=41) (Flaxman et al., 2021 “Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002).” Lancet 398, 981-990) or the Pfizer-BioNtech vaccine BNT162b2 (n=22) (Cele et al., 2021; “Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization”. Nature 602, 654-666e) (Figure 17). For AZD1222, the neutralization of BA.2.75 was 1.2 times lower compared to BA.2 (p=0.0182) and 1.1 times lower compared to BA.2.12.1 (p=0.0065), but increased 1.5 times compared to BA.4 / 5 (p<0.0001) (Figure 17B). Overall, the BA.2.75 neutralizing titer of vaccine serum was lower compared to BA.2, but not to the level seen in BA.4 / 5.

[0298] Vaccine Breakthrough: Neutralization of BA2.75 by serum from BA.1 or BA.2 infection Breakthrough BA.1 serum samples were collected from volunteers who received vaccination at least 28 days after symptom onset (median 38 days, n=16). Pseudovirus neutralization assays were performed on the above panel of pseudoviruses (Figure 17C). The neutralizing titer of BA.2.75 was similar to that of BA.2, 1.4 times (p=0.0052) and 2.0 times (p=0.0001) higher than BA.2.12.1 and BA.4 / 5, respectively, suggesting that BA.2.75 is less likely to cause reinfection in individuals who have suffered a breakthrough BA.1 infection than BA.2.12.1 or BA.4 / 5.

[0299] Breakthrough BA.2 serum samples were collected from vaccinated volunteers at least 12 days after symptom onset (median 29 days, n=23). Pseudoviral neutralization assays were performed against a panel of pseudovirals: Victoria, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75 (Figure 17D). Here, the neutralization titer for BA.2.75 was significantly lower than that for BA.2 (1.4 times, P=0.0021) and similar to that for BA.2.12.1, but still higher than that for BA.4 / 5 (1.4 times, P=0.0123). In summary, BA.2.75 exhibits a certain escape from the humoral response induced by BA.2 breakthrough infection, but not with BA.1 infection.

[0300] Individual BA.2.75 mutations have different effects on neutralization. To understand the effects of individual mutations in BA.2.75 RBD, these mutations were individually introduced into a pseudoviral BA.2 background, and their neutralization was assayed using Pfizer BNT162b2 serum vaccinated three times (Figure 17E). The neutralizing titer of BA.2 decreased in 3 / 4 of the single-mutation variants of BA.2, with the largest decrease being N460K (3.1x, p<0.0001), followed by D339H (1.3x, p=0.0006) and G446S (1.2x, p=0.2312). However, the neutralizing titer increased 1.5 times with the R493Q revertant mutation (p<0.0001). Since Q493 is present in all vaccines, this explains the increased activity of vaccine serum against this revertant mutation.

[0301] Escape from BA.2.75 monoclonal antibody To analyze how BA.2.75 affects neutralizing antibody activity, a pseudoviral assay was used to test a recently reported panel of potent human mAbs generated from cases of omicron breakthrough infection (BA.1 IC50 titer <0.1 μg / ml) (Nutalai et al., 2022) (Figure 19A, Table 22). Of the 27 RBD-specific mAbs, those belonging to the IGHV3-53 / 66 family were most severely affected. Three (Omi-16, Omi-29, and Omi-36) showed complete knockout of BA.2.75 neutralization. Furthermore, four of the four (Omi-18, Omi-20, Omi-27, and Omi-28) showed a more than five-fold decrease compared to BA.2, which is consistent with the observation that N460 interacts with the highly conserved GGS / T motif of CDR-H2 in the structure of the RBD / IGHV-3 / 66 complex (Figure 21B) (Dejnirattisai et al. 2021, Liu et al. 2021, Nutalai et al. 2022).

[0302] Similar to BA.2 and BA.4 / 5, BA.2.75 is not neutralized by the anti-NTD mAb Omi-41, which interacts only with NTDs in BA.1, BA.1.1, and BA.3.

[0303] Omi mAbs were also tested against pseudoviruses encoding the single-point mutation in the BA.2 RBD described above (Figure 23, Table 24). VH3-53 / 66 mAbs that lost neutralization against BA.2.75 were also affected by the N460K mutation, confirming the prediction that this residue is important for binding to several members of this public gene family. Interestingly, the BA.2+N460K mutation alone had a greater effect on the activity of several mAbs than BA.2.75: the neutralization titer of Omi-03 (IGHV3-53) was reduced 50-fold with BA.2+N460K, but only 2-fold with BA.2.75. Omi-17 (IGHV3-66) was completely knocked out with BA.2+N460K, but only 4-fold with BA.2.75. Omi-33 (IGHV3-33) decreased sevenfold in BA.2+N460K, but no change was observed in BA.2.75. Therefore, other mutations in BA.2.75, particularly the R493Q mutation, may have mitigated the effect of the N460K mutation.

[0304] Interestingly, BA.2.75 is more sensitive to Omi-32 (IGHV-3-33) than BA.2, with an eight-fold increase in neutralizing titer. This enhancement of activity by Omi-32 is likely due to a stronger interaction between the antibody and RBD caused by the G446S mutation (Figure 19A, Table 22).

[0305] To confirm that the observed changes in neutralizing activity were related to changes in RBD interaction, binding analysis of selected antibodies to BA.2.75 and BA.2 RBDs was performed by surface plasmon resonance (SPR) (Figure 24). Binding of Omi-29 (IGHV3-53) and Omi-36 (IGHV3-66) to BA.2.75 was severely impaired, while Omi-18 and Omi-20 showed an 8-fold decrease compared to BA.2. On the other hand, the binding affinity of Omi-32 to BA.2.75 was 2-fold increased compared to BA.2, which is consistent with the observed increase in neutralizing titer.

[0306] Escape from commercial monoclonals for BA.2.75 The sensitivity of a panel of mAbs developed as therapeutic agents for BA.2.75 was evaluated (Figure 19B, Table 23). While the neutralization profiles were generally similar for BA.2.75 and BA.2, in addition to 6 / 12 mAbs (REGN10933, ADG10, ADG20, ADG30, Ly-CoV555, Ly-CoV16) that had already completely lost neutralizing activity against BA.2, the residual activity of REG10987 against BA.2 (Weinreich et al., 2021, “REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19”. N Engl J Med 384, 238-251) was further knocked out for BA.2.75 by the G446S mutation (Dejnirattisai et al, 2022). Regarding AstraZeneca AZD1061, its activity against BA.2.75 was similar to that against BA.2 (<3-fold decrease). On the other hand, the titer of AZD8895 recovered from 1.333 μg / ml for BA.2 to 0.008 μg / ml for BA.2.75, showing a 167-fold increase in activity. As a result, AZD7442 (a combination of AZD8895 and AZD1061) (Dong et al., 2021, “Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail”. Nature Microbiol.6, 1233-1244) showed similar activity (2-fold decrease) against both BA.2.75 and BA.2. This result can be explained by the structure of the ancestral SARS-CoV-2 RBD / AZD1061 / AZD8895 ternary complex (Dong et al., 2021). G446 is in contact with CDR-L2 Y55 and W56 of AZD1061, and the G446S mutation causes steric collisions (Figure 21D, E). CDR-H2 of AZD8895 is on top of Q493 of RBD and forms a hydrogen bond with it, while the arginine at 493 violently collides with CDR-H2 of the mAb (Figure 21F, G).S309 activity (Sun and Ho, 2020, “Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic.” Antib Ther 3, 246-256) was three-fold increased in BA.2.75 compared to BA.2, suggesting that the D339H mutation in BA.2.75 reduces the impact of the preceding G339D mutation in BA.2 on S309 activity. LY-CoV1404 (bebtelovimab) (Westendorf et al., 2022, “LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.” Cell Rep 39, 110812) is the only mAb in which neutralization is completely retained across all Omicron sublineages.

[0307] Escape from monoclonal antibodies by BA.2, BA.4, and BA.5 sublineages A decrease in mAb activity was also observed in new BA.2, BA.4, and BA.5 sublineages (including BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BQ.1, BQ.1.1, XBB, XBB.1, and XBB.1.5) (Figure 34), with XBB exhibiting the most extreme escape. BA.2.75.2 reduced the activity of all nine IGHV3-53 / 66 mAbs by more than 100-fold, with complete knockout of activity in 5 out of 9. Only one mAb, Omi-42, remained unaffected by all variants. Omi-42 is unusual in that it binds to the posterior left shoulder of the RBD, a region that has not yet been targeted for mutation by the newly emerging set of BA.2 variants (Nutalai et al., 2022), which is likely due to the relatively small number of antibodies that bind to this region.

[0308] Further data can be found in Nutalai, et al. (2022) “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”, Cell 185(12), 2116-2131; Huo et al. (2022) “Humoral responses against SARS-CoV-2 Omicron BA.2.11, BA.2.12.1 and BA.2.13 from vaccine and BA.1 serum”, Cell discovery 8, 119; and Huo, et al. (2022) “A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75”, Cell Reports, 42(1), 2023.

[0309] Neutralization of BA.2 subtypes BA.2.11, BA.2.12, and BA.2.13 by vaccine serum. The receptor binding capabilities of BA.2 subvariants BA.2.11, BA.2.12, and BA.2.13 were also evaluated. High-resolution crystal structures of BA.2.12.1 RBD were generated, and the novel BA.2 subvariants BA.2.11, BA.2.12, and BA.2.13 showed different sensitivities to serum samples and monoclonal antibodies (mAbs) compared to BA.2.

[0310] Considering the physicochemical properties of the side chain at residue 452, BA.2.13 is expected to undergo a relatively mild change, with the side chain size increasing from L to M while maintaining hydrophobicity. The L to Q transition in BA.2.12.1 introduces a certain polarity, while BA.2.11 is the most radical, with the L to R transition introducing a large basic amino acid.

[0311] Neutralization of BA.2 subtypes BA.2.11, BA.2.12, and BA.2.13 by vaccine serum. To assess the susceptibility of BA.2 subvariants to neutralization by immunoserum, neutralization assays were performed using a series of serum samples with pseudotyped lentiviruses expressing the spike genes of BA.2.11, BA.2.12, and BA.2.13.

[0312] First, we observed the neutralization profiles using serum collected 4 weeks after the third dose of either the Oxford-AstraZeneca vaccine AZD1222 (n=41) or the Pfizer-BioNtech vaccine BNT162b2 (n=18). No significant loss of neutralizing titer was observed compared to BA.2. In fact, BA.2.13 showed a significant increase (1.6 times, p<0.0001) in AZD1222 vaccinated individuals (Figure 27a, b). This contrasts with a recent report (Cao, Y., et al., “BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection”. Nature, 2022) in which serum collected from individuals who had received three doses of the vaccine (four weeks after the third dose of the inactivated CoronaVac vaccine or after the ZF2001 booster dose following the second dose of CoronaVac) showed a significant decrease in neutralizing titers for both BA.2.12.1 and BA.2.13 (BA.2.11 was not tested).

[0313] Vaccine Breakthrough: Neutralization of BA.2 subvariants BA.2.11, BA.2.12, and BA.2.13 by serum from BA.1 or BA.2 infection. Next, we examined the neutralization profiles of serum samples collected from vaccinated individuals infected with BA.1. Samples (n=14) were collected at least 28 days after symptom onset (median 38 days). All individuals in the recovery phase had received at least two doses of the vaccine, and three of them received a third dose after omicron infection. For all three variants, the neutralization titer was significantly lower compared to BA.2, with the greatest decrease being in BA.2.11 (1.6 times, P=0.0067), followed by BA.2.12.1 (1.4 times, P=0.0085) and BA.2.13 (1.2 times, P=0.0085) (Figure 27c). Taken together, these observations suggest that, compared to BA.2, its subvariants do not exhibit stronger humoral immune escape in individuals vaccinated with three doses of AZD1222 or BNT162b2. However, in vaccinated individuals who experience BA.1 breakthrough infection, regardless of the type of vaccine administered, a broad neutralizing antibody response with high titers is induced against all variants of concern, although the BA.2 variant is more adept at evading humoral responses (Nutalai, R., et al., “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”. Cell, 2022. 185(12): p.2116-2131 e18). This may suggest different selective pressures against BA.2 and its subvariants in the high background of breakthrough infection. Since antibody titers naturally decay over longer periods, individuals infected with BA.1 breakthrough are expected to be more susceptible to reinfection with BA.2 subvariants.

[0314] To further elucidate the different reactions between BA.2 and its subvariants, a pseudoviral assay was performed with a panel of potent human monoclonal antibodies (mAbs) generated from cases of BA.1 breakthrough infection (Nutalai, et al., 2022) (Figure 29). Consistent with structural observations and neutralization results, the greatest decrease in neutralizing titer was observed in BA.2.11, followed by BA.2.12.1 and BA.2.13, with BA.2.11 being completely knocked out by 5 / 27 mAbs (Omi-06, Omi-24, Omi-30, Omi-31, and Omi-34). Neutralizing activity against BA.2.12.1 also decreased to varying degrees for the same set of mAbs, while the profile against BA.2.13 remained largely unchanged. Of these, Omi-06 belongs to the IGVH4-4 family, and the other four mAbs belong to the IGVH1-69 family. In fact, previous structural studies had predicted that Omi-06 and Omi-31 would be sensitive to the delta L452R mutation (Nutalai, et al., 2022). To confirm that the observed different neutralizing effects were directly attributable to the changes in RBD binding, surface plasmon resonance (SPR) was used to compare the binding behavior of BA.2 and BA.2.12.1 RBD, using Omi-06 and Omi-31 as examples. As expected, affinity decreased, with BA.2.12.1 RBD binding to Omi-06 being 15 times weaker than BA.2, and significantly, BA.2.12.1 RBD binding to Omi-31 being approximately 1300 times weaker (Figure 30).

[0315] Spike mutations in the BA.2.11, BA.2.12, and BA.2.13 variants may make them slightly more transmissible than BA.2. However, compared to BA.2, healthy vaccinated individuals who received three doses of Oxford-AstraZeneca or Pfizer-BioNtech BNT162b2 vaccine did not appear to gain increased humoral immune escape. This result differs from that observed in vaccinated individuals who received three doses of CoronaVac vaccine, where a significant decrease in neutralizing titer was observed (Cao, Y., et al., BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature, 2022). Nevertheless, vaccinated individuals who experienced BA.1 breakthrough infection showed a significant decrease in neutralizing titers, regardless of the type of vaccine administered. This may be partly due to the partial or complete knockout of the neutralizing activity of antibodies belonging to the IGVH1-69 family, many of which are sensitive to the leucine 452 mutation in the spike RBD. This suggests that the continuously evolving Omicron sublineage may be able to evade the humoral immune response initiated by BA.1, and therefore, in the development of next-generation SARS-CoV-2 vaccines, the BA.1 spike or RBD may not be a significantly superior immunogen to the ancestral Wuhan strain.

[0316] BA.2.12.1 Crystal Structure The crystal structure of BA.2.12.1RBD was measured at 2.38 Å as a ternary complex of neutralizing Fab and nanobody (Figure 27h~m), and it was shown that the structural differences are basically limited to the side chain of residue 452.

[0317] Neutralization of BA.2.75.2 by mAbs produced after BA.1 infection. The neutralization of BA.2.75.2 by an mAb panel constructed after BA.1 infection (Nutalai et al., 2022) was investigated. A decrease in mAb activity against BA.2.75.2 was observed (Table 32a). BA.2.75.2 reduced the activity of all nine IGVH3-53 / 66 mAbs by more than 100-fold, with complete knockout of activity in 4 out of 9. Only one mAb, Omi-42, remained unaffected by all variants and showed neutralization of the BA.4+14 mutation as described by an IC50 of 11 ng / ml. Omi-42 is unusual in that it binds to the posterior left shoulder of the RBD, a region that has not yet been targeted by the mutation (Nutalai et al., 2022), which is likely due to the relatively small number of antibodies that bind to this region.

[0318] mAb panels developed for clinical use were also tested (Dong et al., 2021; Sun and Ho, 2020; Weinreich et al., 2021; Yuan et al., 2022). Many of these were significantly affected by several variants. The activity of all mAbs except S309 was knocked out by one or more variants, including Ly-CoV1404 (Westendorf et al., 2022) (see Table 32b).

[0319] Serum neutralizing titers of Pfizer BNT162b2 vaccine against BA.2.75.2 and BA.2.3.20. In the neutralization of serum collected 28 days after the third dose of the Pfizer BNT162b2 vaccine (Polack et al., 2020) and in cases of BA.4 / 5 infection with BA.1 and BA.2, the characteristics of these subjects are described in the methods.

[0320] When serum obtained 28 days after BNT162b was used, the infectivity titer against BA.2.75.2 was significantly lower compared to BA.2 and BA.4, and was the lowest among all variants tested compared to the ancestral Victoria strain. The decrease in titer against BA.2.75.2 was in contrast to BA.2.75, which showed only a slight decrease compared to BA.2. The titer against BA.2.3.20 was also significantly lower.

[0321] When serum obtained after BA.1, BA.2, and BA.4 / 5 infection was used, the neutralizing titers of BA.2.75.2 and BA.2.3.20 were similarly significantly lower compared to BA.4 / 5. The neutralization of the BA.4+14 RBD mutation also decreased compared to BA.2 and BA.4 / 5, but not significantly more than that of BA.2.75.2, indicating the dominant influence of the BA.2.75.2 mutation.

[0322] Vaccine Breakthrough: Neutralization of BA4.6 by serum from BA.1 or BA.2 infection Here, the inventors study the neutralization profile of BA.4.6 using: Pfizer-BioNtech vaccine serum, BA.1, BA.2, and BA.4 / 5 vaccine breakthrough immunizing serum, and a panel of monoclonal antibodies. Notably, the inventors demonstrate further antibody avoidance of BA.4.6, providing guidance for vaccine design and the use of therapeutic monoclonal antibodies.

[0323] To evaluate the antibody evasion ability of BA.4.6, a panel of pseudotyped lentiviruses expressing the S gene from BA.4.6 and other SARS-CoV-2 variants (Di Genova, C., et al., Production, titration, neutralization and storage of SARS-CoV-2 lentiviral pseudotypes. figshare, 2020.) was constructed, along with Victoria, an early Wuhan-related strain of the pandemic, used as a control. First, the neutralization profile of serum collected 4 weeks after the third dose of the Pfizer-BioNtech vaccine BNT162b2 was examined (n=22). Compared to BA.4 / 5, the neutralizing titer against BA.4.6 was twice as low in BNT162b2 serum (p<0.0001) (Figure 28a).

[0324] The neutralization profiles of serum samples collected from vaccinated individuals infected with BA.1 were assayed. Samples (n=16) were collected more than 28 days after symptom onset. BA.2 samples (n=23) were collected more than 12 days after symptom onset or in BA.4 / 5 cases. Samples (n=11; all but one were vaccinated) were collected more than 23 days after symptom onset (Figures 28b-d). The neutralization titer against BA.4.6 was significantly lower for both breakthrough BA.1 (1.5 times, P=0.0006) and BA.2 (1.2 times, P=0.0384) serum samples compared to BA.4 / 5. Notably, BA.4.6 was able to effectively escape neutralization by serum samples from BA.1 breakthrough infection, with significantly reduced titers compared to BA.1 (4.4-fold, p=0.0001), BA.2 (3-fold, p=0.0009), and BA.4 / 5 (1.5-fold, p=0.0006). In the BA.4 / 5 breakthrough cohort, a slight, non-significant increase in neutralization titer for BA.4.6 was observed compared to BA.4 / 5.

[0325] To further characterize the antigen escape properties of BA.4.6, a pseudoviral assay was performed with a panel of potent human mAbs generated from BA.1 breakthrough recoveries (Nutalai et al., 2022) (Figure 28e). In general, the neutralization profile of BA.4.6 was similar to that of BA.4 / 5. However, the residual activity of Omi-35 (IC50 = 1.687 μg / mL) was further knocked out for BA.4.6, and the potency of Omi-32 and Omi-33 against BA.4 / 5 (IC50 = 0.035 and 0.013 μg / mL, respectively) was completely lost for BA.4.6. The loss of Omi-32 activity can be explained by the disruption of the interaction between H1 and R346, as shown in previous structural analyses (Nutalai et al., 2022).

[0326] Neutralization of BA.4.6 by mAbs in clinical use Finally, the neutralizing activity of several mAbs in clinical use was evaluated (Figure 28f). The efficacy of AZ1061 / silgavimab against BA.4 / 5 was completely knocked out against BA.4.6, and AZ7742 / Evasheld (a combination of AZ1061 / silgavimab and AZ8895 / thixagevimab, which is already inactive against BA.4 / 5) resulted in a complete loss of activity. The activity of S309 / sotorobimab (which, due to its ineffectiveness against BA.2, is no longer approved by the U.S. Food and Drug Administration (FDA) for the treatment of COVID-19 as of April 2022) was further reduced compared to BA.2 and BA.4 / 5. Therefore, Ly-Cov1404 / bebuterobimab remains the only treatment option for BA.4.6.

[0327] In summary, BA.4.6 was shown to be even more resistant to serological neutralization from individuals who had received three doses of Pfizer vaccine and from individuals who had recovered from BA.1 and BA.2 vaccine breakthroughs, compared to BA.4 / 5. Notably, BA.4.6 does not appear to be more resistant to serological neutralization from BA.4 / 5 breakthrough infections compared to other variants. Taken together, these findings suggest that unless an individual has recovered from a BA.4 / 5 infection that would provide some protection against BA.4.6 and has received three doses of vaccine, they are likely to develop an infection or breakthrough infection with BA.4.6.

[0328] As of September 2022, a bivalent booster vaccine combining the ancestral strain and Omicron BA.1 is being deployed in the UK and was recently approved by the FDA. The extent to which these bivalent boosters are effective in preventing BA.4.6 infection remains unknown. Finally, BA.4.6 further impaired the activity of Evasheld, which had maintained activity against BA.4 / 5. As a result, currently, only LY-CoV1404 bebuterovimab retains efficacy against all circulating SARS-CoV-2 variants.

[0329] Systematic themes in mAb interactions Omi-3 (representative of the IGVH3-53 gene family) and AZD8895 (IGVH1-58) both contact F486. The F486V mutation has little effect on Omi-3 (Figures 10F, G, 11F), but the neutralization of AZD8895 and other IGVH1-58 mAbs (e.g., Omi-12) is significantly reduced (Figures 10D, E, 11E). Notably, numerous Omi series antibodies (total 9 / 28, Figure 8A, Table 21) belonging to the closely related IGVH3-53 and IGVH3-66 gene families are almost completely resistant to BA.4 / 5 changes, while the majority of antibodies from these gene families induced against previous variants are knocked out at BA.1 and BA.2 (Nutalai et al., 2022). This is consistent with the selection of a subset of antibodies that are insensitive to further BA.4 / 5 mutations through breakthrough omicron infection.

[0330] The effects on antibodies with broadly similar epitopes can differ dramatically, and this is also true for antibodies with 452 or 486 at the center of their binding footprint. For example, both Omi-31 (IGVH1-69) and Omi-32 (IGVH3-33) bind in front of the right shoulder, and their CDR-H3 is located near 452, but the activity of Omi-31 is inactivated by L452R (as detailed above), while the activity of Omi-32 is significantly enhanced (Figures 8A, 11A, B). Similarly, both Omi-25 and Omi-42 belong to the IGVH3-9 gene family, and their footprints are in the 486 region (Figures 11C, D). Omi-25 contacts F486, and the neutralization of BA.4 / 5 is suppressed. In contrast, Omi-42 does not come into contact with any of the mutation sites, and neutralization for BA.4 / 5 is completely preserved (Figures 10H, I, 11D).

[0331] Detailed mapping of RBD antibody binding using competitive assays. Using a matrix of pairwise BLI measurements, we mapped strongly RBD-bound omicron mAbs to several pre-pandemic mAbs with known binding sites.

[0332] This method yielded consistent predictions. The mAbs segregate into a limited set of epitopes, which appear to be a subset of the epitopes observed in the early pandemic virus and are quite different from the focus seen in beta. Essentially, the antibodies cluster into two regions: one containing VH3-53 and VH3-66 type antibodies is located on the posterior side of the neck / left shoulder and extends to the upper part of the left shoulder, and the other is located on the anterior side of the neck / right shoulder and extends toward the known antibody binding site of S309. This region is occupied by VH1-69 family antibodies, with the exception of Omi-2, which is located within the other cluster. mAb Omi-09, which shows reduced neutralization in beta and gamma, is located near residue 484 and is mutated from Glu to Lys in beta / gamma and to Ala in omicron. VH1-69 mAb Omi-24, 30, 31, and 34, which show reduced delta neutralization, are located near residue 452, where the delta is mutated from Leu to Arg.

[0333] Structure of the anti-omicron Fab / RBD complex Structural analysis was performed on selected potent omicron mAbs. The crystal structures of complexes between omicron BA.1 RBD and three different Fabs: Omi-3, 9, and 12 were determined. Due to the low resolution (5.5 Å) of the Omi-12 complex, the structure of the Fab alone was determined at high resolution, and the structure of the complex was obtained by rigid body fitting.

[0334] Omi-3 belongs to the VH3-53 gene family and demonstrates how this gene family can adapt to broadly neutralize all major SARS-CoV-2 variants (however, like all potent omicron antibodies, it does not bind to SARS-CoV-1 RBD). The fundamental problem with these antibodies is that most VoCs have the mutation N501Y, which introduces a steric collision with LC CDR1(L1), rendering the binding of antibodies containing the majority of VH3-53 ineffective. However, two mechanisms for moving L1 to avoid this collision have been reported in the past (Dejnirattisai et al., 2021b; Liu et al., 2021b). In mAb-222 isolated from individuals infected with early pandemic strains, a proline insertion at residue 30 allows it to fill Tyr-501 without collision (Dejnirattisai et al., 2021b), thereby effectively neutralizing alpha, beta, and gamma variants. Beta-27 uses an alternative mechanism to extend the HC CDR3(H3) loop from the usual 9 residues to 11 residues, replacing L1 and creating enough space for 501Y to be stabilized by a main chain interaction that confers similar cross-reactivity (Liu et al., 2021b).

[0335] Omi-3 uses the same mechanism as beta-27 to address the N501Y mutation, but the Omi-3 H3 is again one residue longer. Other VH3-53 omicron antibodies (Omi-18 and Omi-29) have H3 very similar to beta-27 and likely use the same mechanism. This L1 configuration is also compatible with the Y505H mutation in omicron. However, neither 222 nor beta-27 can effectively neutralize omicron, which may be due to specific features of the H3 loop that create close contact with the Q493R omicron mutation.

[0336] Omi-9 is one of three VH3-30 mAbs and binds across the left shoulder of RBD. Omi-9 exhibits relatively weak beta and gamma neutralization (Figure 2). Other antibodies with high sequence similarity bind similarly, with H3 in contact with residue 484. The Omi-9 / BA.1 complex has lower resolution (4.2 Å), but it is clear that H3 is in contact with residue 484, which explains the sensitivity to E484K in beta and gamma, while E484A is tolerated in Omicron.

[0337] Omi-12 belongs to the VH1-58 gene family (it is the only member of this family among the 28 potent omicron antibodies). Like Omi-12, several members of this gene family have a glucosylation site at residue 102 of the heavy chain CDR3, but its role is unknown. VH1-58 antibodies induced in the early stages of a pandemic or during betavirus infection show a reduced ability to neutralize omicrons. For example, mAb253, beta47, and known antibody D (AZD8895) each show reduced activity of omicron BA.1 against Victoria.

[0338] In contrast, Omi-12 is adaptive and can potently neutralize omicrons and all VoCs (Figure 2A, B). The VH1-58 antibody binds to the left shoulder epitope, and H3 contacts S477N, but the mutation at this position in iota did not affect the neutralization of VH1-58 mAb using a pseudovirus assay. Furthermore, mAb253 can still neutralize delta despite the T478K mutation. BA.2 of early pandemic mAb150 (VH3-53). Detectable residual activity was observed with BA.1, BA.1.1 and BA.2 (BA.3 was not tested). Two complex structures were obtained in different spatial groups, but these were very similar and provided three independent perspectives on the complex. mAb150 binds in a similar pose to those previously observed for early pandemic viruses, but is translated, forming a looser interaction, consistent with the near complete loss of neutralizing activity. This demonstrates the dramatic impact of adaptive mutations observed in Omi-3.

[0339] Interestingly, in BA.2, the three serine residues mutated in BA.1 RBD—S371L, S373P, and S375F within the loop adjacent to the lipid-binding pocket—are also mutated. However, the mutation at 371 is a mutation to Phe, which is likely a single point mutation from the early stages of the pandemic, whereas the S317L mutation in BA.1 requires two mutations. Therefore, BA.2 may have a function common to earlier versions of the Omicron lineage. Furthermore, the various perspectives provided for this part of the structure indicate the adoption of certain different three-dimensional structures. This is likely because different crystals are in contact, reflecting the flexibility of this loop region. The Ser mutation requires a double codon change and may have a biological function, as it could affect the presentation of the RBD. In the early pandemic virus, VoC, Omicron BA.1, and BA.2, this loop can be observed from multiple perspectives, confirming that flexibility is maintained across all variants.

[0340] Modeling the effects of selected commercially available known antibodies, early pandemic conditions, and beta mAbs on changes in BA.1, BA.1.1, and BA.2. Known antibodies A (REGN10987) and B (10933): Known antibody B (REGN10933) binds behind the left shoulder, and REGN10987 binds to the right shoulder. The activity of both is knocked out by the Omicron strain, separately from known antibody A (REGN10987) by BA.2. Known antibody B (REGN10933)H2 contacts residue 493, and since Q493R is present in all Omicron strains, neutralizing activity against Omicron is universally lost. Known antibody A (REGN10987)H2 contacts residue 446. Since BA.2 lacks the G446S mutation, regn10987 retains some degree of neutralizing ability.

[0341] Known antibodies C (AZD1061) and D (AZD8895): Known antibodies C (AZD1061) and D (AZD8895) bind to the back of the left shoulder and the front of the right shoulder, respectively, and both show a reduced neutralizing effect. Known antibody C (AZD1061) can still neutralize BA.2 and BA.3 (by about 10 times), but the neutralization of BA.1 is reduced by more than 100 times compared to Victoria, and the neutralization of BA.1.1 is reduced by more than 1000 times compared to Victoria. Known antibody C (AZD1061) is affected by contact with the G446S (not present in BA.2 and BA.3) and R346K (BA.1.1) mutations (L2 and H3 in contact). Known antibody D (AZD8895) is a VH1-58 antibody that contacts residues 477 (H3) and 493 (H2) and is impaired by the S477N and Q493R mutations, which are commonly present in the Omicron strain. Known antibody E (AZD7442) (a combination of C and D), as a whole, maintains some degree of neutralizing activity against the Omicron strain.

[0342] Known antibodies F, G, and H: The activity of all known antibodies F, G, and H against omicron is significantly reduced. The activity of known antibodies F and H is completely lost, and the activity of known antibody G (ADG20) against omicron is reduced by 276 times.

[0343] Known antibodies I and J: The activity of both antibodies is knocked out across the entire Omicron lineage. Known antibody J (Ly-CoV16) (VH3-53) interacts extensively with N501 and Y505 via L1 and L3, making it sensitive to mutations at these residues. Known antibody I (Ly-CoV-555) is vulnerable to the E484K mutation in the delta but appears to be resistant to E484A. However, because it also contacts residue 493, the universal Omicron Q493R mutation renders the binding throughout the antibody ineffective.

[0344] Known antibody K(S309): Known antibody K(S309) maintains appropriate activity across the entire Omicron lineage. S309 binds to H3 on the right side and contacts G339 and N343 glycans. The latter is close to serine 371, 373, and 375 mutations. The S371F mutation in BA.2 (in contrast to S371L) affects binding and may slightly weaken activity against this virus.

[0345] BA.2RBD structure and ACE2 affinity The affinity of Omicron BA.1, BA.1.1, BA.2, and BA.3 RBDs for ACE2 was measured by SPR and BLI. The affinity of BA.1 was comparable to that of the initial virus, at 8nM and 7nM, respectively (binding affinities of Omicron RBDs are shown in Tables 14 and 18). This indicates that the increase in affinity brought about by S477N, Q498R, and N501Y is offset by other mutations in the ACE2 footprint. The affinity of BA.2 was slightly increased compared to the initial virus (approximately 1.5 times for xnM and YnM, respectively). Based on previous measurements of the effect of individual mutations on binding affinity, G496S and the triple mutations S371L, S373P, and S375F reduce binding by 2x and 2.2x, respectively, while BA.2 lacks G496S and has S371F. This may explain some of the differences, but it is more likely that the mutations in BA.2 at the edge of the ACE2 footprint enhance binding. This is confirmed by the structure of BA.2 / ACE2.

[0346] Affinity between BA.4 / 5 RBD and ACE2 The affinity of BA.4 / 5RBD for ACE2 was also measured by SPR (Figure 12A-D). The affinity of BA.4 / 5RBD increased compared to the ancestral virus (Wuhan), BA.1, and BA.2 (approximately 3 times, 3 times, and 2 times, respectively (BA.4 / 5 / ACE2 KD=2.4nM) (Dejnirattisai et al., 2022; Nutalai et al., 2022)). This is mainly due to an increase in binding half-life. Modeling of the ACE2 / RBD complex suggests that the majority of this effect arises from electrostatic complementarity between ACE2 and RBD brought about by the L452R mutation (Figure 12E-G).

[0347] Affinity between BA.2.75RBD and ACE2 Surface plasmon resonance (SPR) was also used to characterize the interaction between ACE2 and BA.2.75 RBD. The dissociation rate was very slow, resulting in a sub-nanomolecular affinity (BA.2.75 / ACE2 KD = 0.45 nM) (Figure 18A, B). This represents a significant increase in affinity compared to BA.2 (9x) (Figure 18C), and is even stronger than BA.4 / 5 (5x) (Figure 18D), which binds to ACE2 with a higher affinity than BA.2 (Tuekprakhon et al., 2022). BA.2.75 was found to be the most potent ACE2 binder among all SARS-CoV-2 VoCs, including alpha (alpha / ACE2 KD = 1.5 nM, Figure 18E), and the first SARS-CoV-2 VoC to exhibit sub-nanomolecular affinity.

[0348] Although BA.2+N460K RBD could not be expressed, the binding affinity of BA.2+R493Q RBD to ACE2 (Figure 18F) was also measured (KD = 0.55 nM). This confirms that the R493Q revertant mutation contributes to the high affinity of BA.2.75 RBD.

[0349] The effects of mutations in BA.2.75 A series of mutations in BA.2.75, compared to BA.2, have opposing effects on neutralization. The revertant mutation R493Q facilitates neutralization of the virus using vaccine serum (the vaccine contains Q493), while N460K, when expressed alone, significantly reduces the neutralizing titer compared to the combination of mutations found in BA.2.75. N460K is a novel substitution not seen in previous variants of SARS-CoV-2. This mutation was introduced into the BA.2 backbone, and its effect on neutralization with BNT162b2 serum was evaluated. Notably, the BA.2+N460K titer was 3.1 times lower than that of BA.2, which was greater than the decrease seen in BA.2.75 and comparable to the decrease seen in BA.4 / 5.

[0350] Using a panel of potent mAbs derived from vaccinated individuals who underwent BA.1 vaccine breakthrough infection, it was shown that several mAbs belonging to the IGHV3-53 / 66 family exhibited reduced or knocked-out activity against BA.2.75. IGHV3-53 / 66 is the most frequently isolated mAb in SARS-CoV-2 and binds to the “neck” epitope. It is not surprising that IGHV53 / 66 thus forms the major public antibody response, and that the virus has evolved to escape this response.

[0351] Although BA.2+N460K RBD could not be expressed, previous studies using yeast displays have shown that N460K can enhance ACE2 RBD binding, an effect similar to that seen with the N501Y mutation, which was first described in alpha (Zahradnik et al., 2021). Therefore, N460K may enhance antibody escape and increase receptor binding affinity.

[0352] Interestingly, BA.2.75 also acquired the R493Q reversion (Q493R was acquired in BA.1 and is present in all other Omicron sublineages except BA.4 / 5). BA.2.75RBD was able to bind to ACE2 with 9 times higher affinity than BA.2 and more strongly than BA.4 / 5 (Dejnirattisai et al., 2022; Tuekprakhon et al., 2022). This is partially caused by the R493Q mutation. BA.2.75RBD has the highest receptor binding affinity of all SARS-CoV-2 variants measured to date.

[0353] These data suggest a delicate balance may exist between antibody escape and ACE2 receptor affinity. Mutations in BA.2.75 reduce the neutralizing titer in vaccine serum compared to BA.2. Individual mutations in BA.2.75 can cause a significant decrease in neutralizing titer compared to the complete BA.2.75S sequence, but these are balanced by the R393Q revertant mutation, which may have been selected to increase affinity for ACE2 and enhance the transmissibility of BA.2.75.

[0354] Affinity between BA.2.11, BA.2.12, and BA.2.13RBD and ACE2 To assess the potential changes in the transmissibility of BA.2 subvariants, SPR experiments were performed to analyze RBD binding to ACE2 (Figure 27d-g). The three RBD variants had an affinity of approximately 3 nM to ACE2, which was slightly higher than that of the previously reported BA.2 RBD (KD=4 nM) (Nutalai et al., 2022). Modeling of the ACE2 / RBD complex suggests that this increase in affinity may result from a slight improvement in complementarity between ACE2 and RBD due to the leucine 452 mutation. Therefore, these variants may have a slight advantage in transduction over BA.2.

[0355] Antigen maps of BA.3 and BA.4 / 5 The neutralization data described above were used to position BA.3 and BA.4 / 5 on the antigen map. The methods used for delta and omicron variant analysis were repeated (Liu et al., 2021, “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum”. Cell 184, 4220-4236 e4213), where individual viruses were modeled independently, allowing for serum-specific scaling of the response. The measured and modeled response is shown in Figure 13A (1551 observations and 340 parameters, residual error 23%). The results are best visualized in three dimensions (see the 2D projection in Figure 13B). This indicates, as expected, that the omicron sublineages cluster together but are well isolated from the early pandemic virus and early VoC. Within the omicron cluster, BA.4 / 5 is furthest from the preomicron virus.

[0356] Antigen map of BA.2.75 Neutralization of BA.2.75 was tested using serum from individuals previously infected during the course of the pandemic. These included serum obtained in the early stages of the pandemic (before the emergence of alpha), along with serum obtained after infection with alpha, beta, gamma, delta, BA.1, and BA.2 (Figure 25). As expected, the BA.2.75 neutralizing titer was lower than that of homologous infection strains (e.g., alpha serum against alpha virus). However, most notably, there was a complete loss of BA.2.75 neutralization when using delta serum (no samples achieved 50% neutralization at a 1 / 20 dilution). However, titers against BA.2.75 were much higher in cases vaccinated before or after delta infection.

[0357] Using these data, BA.2.75 was plotted on a 3D antigen map using the method previously reported by Tuekprakhon et al., 2022 (Figure 22A, B). All VoCs were initially included (Figure 22A). This showed that BA.2.75 was grouped with other omicron viruses and isolated in one hemisphere of the 3D plot. BA.2.75 appeared to be well isolated from other omicron sublineages, particularly BA.4 / 5. It is also noteworthy that BA.2.75 and Delta are located diametrically opposite each other in the figure, highlighting the antigenic distance between these two viruses. Because the data are high-dimensional, true distances may be distorted in 3D projection, and therefore only omicron and early pandemic viruses were calculated (however, their complete serological information is retained). The results are shown in Figure 22B, which reproduces the main features of the complete plot, but allows for a wider distribution of omicron sublineages in 3D space. Notably, when clustered in the early stages of the pandemic and the fusion of BA.2 / BA.3 pairs, the points are distributed as a bicornuate pyramid that maximizes segregation, consistent with antigen escape being a key evolutionary factor.

[0358] Example 3. An example of an antibody that can be produced by exchanging light chains between antibodies derived from the same heavy chain V-gene. As discussed in the detailed explanation above, antibodies derived from the same heavy chain V gene may exchange their light chains to produce an antibody containing the heavy chain variable region of the first antibody and the light chain variable region of the second antibody, and such a new antibody may have improved neutralization and / or other characteristics compared to the “parent” antibody.

[0359] Tables 4-12 provide examples of antibodies that can be produced by exchanging the light chains between antibodies derived from the same heavy chain V gene. Table 17 provides information on the heavy and light chain V genes from which 28 omicron-specific mAbs are derived, along with their specificity for RBD or NTD of the SARS-CoV-2 spike protein.

[0360] Example 4. Materials and Methods virus strain SARS-CoV-2 / Human / AUS / VIC01 / 2020 (Caly et al, 2020), alpha and beta were provided by Public Health UK, gamma was cultured from pharyngeal swabs from Brazil, delta was donated from the UK G2P genotype phenotyping consortium by Wendy Barclay and Thushan de Silva, and omicron was cultured from positive pharyngeal swabs (IRAS Project ID: 269573, Ethics Ref: 19 / NW / 0730). Briefly, VeroE6 / TMPRSS2 cells (NIBSCs) were cultured in 1% fetal bovine serum, 2 mM Cells were maintained at 37°C in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with Glutamax, 100 IU / ml penicillin-streptomycin, and 2.5 ug / ml amphotericin B, in the presence of 5% CO2, and then inoculated with 200 ul of swab solution. Cells were further maintained at 37°C, and cytopathic effects (CPE) were observed daily. Virus-containing supernatants were clarified with 80% CPE by centrifugation at 3,000 rpm at 4°C and then stored at -80°C in single-use aliquots. Viral titers were measured by a focus formation assay in Vero CCL-81 cells (ATCC).

[0361] Sequencing of the Omicron isolate revealed expected consensus S gene changes (A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), intact furin cleavage sites, and a single additional mutation, A701V.

[0362] Cells were infected with the SARS-CoV-2 virus using an MOI of 0.0001.

[0363] The supernatant containing the virus was collected at 80% CPE, centrifuged at 4°C and 3000 rpm, and then stored at -80°C. Viral titers were measured by a focus formation assay in Vero cells. The Victoria passage 5, Alpha passage 2, and Beta passage 4 strains, as well as the Gamma passage 1, Delta passage 3, and Omicron passage 1 strains, were sequenced to verify that there were no expected changes in the spike protein sequence and furin cleavage sites.

[0364] Bacterial strains and cell cultures Vero (ATCC CCL-81) and Vero E6 / TMPRSS2 cells were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX (Gibco, 35050061), and 100 U / ml penicillin-streptomycin. Human mAbs were expressed in HEK293T cells cultured at 37°C and 5% CO2 in UltraDOMA PF protein-free medium (Cat#12-727F, LONZA). HEK293T (ATCC CRL-11268) cells were cultured at 37°C and 5% CO2 in DMEM high glucose (Sigma-Aldrich) supplemented with 10% FBS, 1% 100X Mem Neaa (Gibco), and 1% 100X L-glutamine (Gibco). RBD, RBD variant, and ACE 2 To express the plasmid, HEK293T cells were cultured at 37°C in DMEM high glucose (Sigma) supplemented with 2% FBS, 1% 100X Mem Neaa, and 1% 100X L-glutamine for transfusion. Omicron RBD and human mAbs were also expressed in HEK293T (ATCC CRL-11268) cells cultured at 37°C and 5% CO2 in FreeStyle 293 Expression Medium (ThermoFisher, 12338018). Escherichia coli (E. coli) DH5α bacteria were used for plasmid transformation and large-scale preparation. Single colonies were selected and cultured overnight at 37°C and 200 rpm in LB broth in a shaker.

[0365] Serum from Pfizer vaccine recipients Pfizer vaccine serum was obtained from volunteers who had received one or two doses of the BNT162b2 vaccine. Vaccinators were healthcare workers based at the Oxford University Hospitals NHS Foundation Trust who were not known to have previously been infected with SARS-CoV-2 and were enrolled in the OPTIC trial as part of Oxford Translational Gastrointestinal Unit GI Biobank Study 16 / YH / 0247 [Yorkshire and Humber-Sheffield Research Ethics Committee (REC)], amended for this purpose on June 8, 2020. The trial was conducted in accordance with the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines for conducting clinical trials of medicinal products. Written informed consent was obtained for all participants enrolled in the trial. Participants received intramuscular administration of 30 micrograms of Pfizer / BioNtech BNT162b2 mRNA vaccine, diluted (0.3 mL each), at intervals of 17–28 days. Samples were then collected approximately 28 days (range 25–38), 180 days (range 178–221), and 270 days (range 243–273), followed by a third "booster dose" of the BNT162B2 vaccine, approximately 28 days (range 25–56). The average age of vaccinated individuals was 37 years (range 22–66), with 21 males and 35 females.

[0366] Plasma from early and alpha cases of the pandemic Participants from the first wave of SARS-CoV-2 in the UK and those whose B.1.1.7 lineage sequences were confirmed in December 2020 and February 2021 were recruited through three studies: Sepsis Immunomics [Oxford REC C, reference: 19 / SC / 0296], ISARIC / WHO Clinical Characterization Protocol for Severe Emerging Infections [Oxford REC C, reference: 13 / SC / 0149], and Gastrointestinal Diseases in Oxford: COVID Sub-Study [Sheffield REC, reference: 16 / YH / 0247]. Diagnosis was confirmed by reporting of symptoms consistent with COVID-19 and by testing positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from upper respiratory tract (nasal / pharyngeal) swabs tested at an accredited laboratory. Blood samples were collected with consent at least 14 days after symptom onset. Clinical information, including disease severity (mild, severe, or critical infection according to World Health Organization recommendations), the time between symptom onset and sample collection, and the participant's age were obtained for all individuals at the time of sample collection. After thermal inactivation, plasma / serum samples were divided into equal parts so that no more than three freeze-thaw cycles could be performed for data generation.

[0367] Serum from Beta, Gamma, Delta, and BA.1 infection cases Beta and delta samples from UK infection cases were collected according to the Oxford Gastro-intestinal illness protocol, "Innate and adaptive immunity against SARS-CoV-2 in healthcare worker family and household members": a COVID sub-study previously discussed and approved by the University of Oxford Central University Research Ethics Committee. All individuals had sequenced beta / delta infection or PCR-confirmed symptomatic illness. These were present during isolation and in direct contact with sequenced beta / delta cases. Additional beta-infected serum (sequenced) was obtained from South Africa. At the time of swab collection, patients signed informed consent agreeing to the collection of data and serial blood samples. The study was approved by the University of the Witwatersrand Human Research Ethics Committee (reference number 200313) and conducted in accordance with Good Clinical Practice guidelines. As part of a nationwide investigation for coronavirus, gamma samples were provided by the International Standard Laboratory for Coronavirus (WHO) at FIOCRUZ, and approval was obtained from the FIOCRUZ Ethics Committee (CEP 4.128.241) to sequentially accept and analyze samples from suspected COVID-19 cases for virological investigation. Clinical samples were shared with Oxford University, UK under MTA IOC FIOCRUZ 21-02.

[0368] BA.1 infection cases, serum from study subjects Following informed consent, individuals with Omicron BA.1 were concurrently enrolled in the ISARIC / WHO Clinical Characterization Protocol for Severe Emerging Infections [Oxford REC C, rec. 13 / SC / 0149] and the Oxford Gastro-intestinal illness protocol, “Innate and adaptive immunity against SARS-CoV-2 in healthcare worker family and household members” protocol: a COVID sub-study further approved by the University of Oxford Central University Research Ethics Committee [Sheffield REC, rec. 16 / YH / 0247]. Diagnosis was confirmed by reporting symptoms consistent with COVID-19 or positive contact with a known Omicron-infected individual, and by positive testing for SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) from upper respiratory tract (nasal / throat) swabs tested in an accredited laboratory, and by lineage sequencing confirmed by a national reference laboratory. Blood samples were collected with consent at least 10 days after confirmation of PCR testing. Clinical information, including the severity of the disease (mild, severe, or critical infection according to recommendations from the World Health Organization), the time between symptom onset and sample collection, and the age of the participants were obtained for all individuals at the time of sample collection.

[0369] Astrazeneca-Oxford vaccine testing procedure and sample preparation. Full details of the randomized controlled trials of ChAdOx1 nCoV-19 (AZD1222) were previously published (PMID:33220855 / PMID:32702298). These trials were registered with ISRCTN (15281137 and 89951424) and ClinicalTrials.gov (NCT04324606 and NCT04400838). Copies of the protocols were included in previous publications (Folegatti et al., 2020, Lancet 396, 467-478).

[0370] Data from vaccine volunteers who received two doses of the vaccine are included in this example. The vaccine dose was 5 × 10 10 The dose was either one viral particle (standard dose; SD / SD cohort n=21) or half the dose (low dose) as the first dose and the standard dose as the second dose (LD / SD cohort n=4). The interval between the first and second doses ranged from 8 to 14 weeks. Blood samples were collected on the day of vaccination and on pre-specified days after vaccination, e.g., 14 and 28 days after booster immunization, and serum was separated.

[0371] Focus reduction neutralization assay (FRNT) The neutralizing potential of Ab was measured using a focus reduction neutralization test (FRNT), and the reduction in the number of infected foci was compared to a negative control well without antibody. Briefly, serially diluted Ab or plasma was mixed with the SARS-CoV-2 strain and incubated at 37°C for 1 hour. The mixture was then transferred to a 96-well, cell culture-treated flat-bottom microplate containing two confluent Vero cell monolayers and incubated for a further 2 hours, after which 1.5% semi-solid carboxymethylcellulose (CMC) overlay medium was added to each well to restrict viral spread. Next, the focus formation assay was performed by staining Vero cells with human anti-NP mAb (mAb206) followed by goat anti-human IgG (A0170; Sigma) conjugated to peroxidase. Finally, approximately 100 foci (infected cells) per well were visualized in the absence of antibody by adding TrueBlue peroxidase substrate. Virus-infected cell foci were counted using the classic AID EliSpot reader with AID EliSpot software. The percentage of focus reduction was calculated, and IC50 was determined using the probit program in the SPSS package.

[0372] Plasmid construction and pseudotyped lentiviral particle generation Pseudotyped lentiviruses expressing the SARS-CoV-2 S protein from the ancestral strain (Victoria, S247R), BA.1, BA.1.1, and BA.2 were constructed as previously described (Nie, Jianhui, et al. "Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2." Emerging microbes & infections 9.1(2020):680-686., Liu, Chang, et al. "Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum." Cell 184.16(2021):4220-4236.), with several modifications. Briefly, synthetic codon-optimized SARS-CoV-2 BA.1 and BA.2 were custom synthesized by GeneArt (Thermo Fisher Scientific GENEART). Insertion fragments and pcDNA3.1 vectors were cloned using Gibson assembly. The Victoria (S247R) construct is as previously described in Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16(2021):4220-4236.To construct BA.1.1, the BA.1 construct was used as a template, and mutagenic primers for R346K (R346K_F 5'-GTGTTCAATGCCACCAAATTCGCCAGCGTGTAC-3' and R346K_R5'-GTACACGCTGGCGAATTTGGTGGCATTGAACAC-3') were PCR amplified together with two primers for the pcDNA3.1 vector (pcDNA3.1_BamHI_F 5'-GGATCCATGTTCCTGCTGACCACCAAGAG-3' and pcDNA3.1_Tag_S_EcoRI_R 5'-GAATTCTCACTTCTCGAACTGAGGGTGGC-3'). The constructs were purified using the QIAquick Gel Extraction Kit (QIAGEN) and Gibson assembly was performed. All constructs were validated by Sanger sequencing after plasmid isolation using the QIAGEN Miniprep Kit (QIAGEN).

[0373] A similar strategy was applied to BA.3 and BA.4 / 5, and in short, the BA.3 mutation was constructed using combined fragments of BA.1 and BA.2. The resulting mutations are as follows: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211 / L212I, G339D, S371F, S373P, S375F, D405N, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. The BA.4 / 5S protein shared several amino acid mutations with BA.2 (Nutalai et al., 2022), but Δ69-70, L452R, F486V, and R498Q were added to generate the BA.4 / 5 mutation. The resulting pcDNA3.1 containing the S gene was used to generate pseudoviral particles along with a lentiviral packaging vector and a transfer vector encoding a luciferase reporter. Structural integrity was confirmed by sequencing.

[0374] Using the same method, BA.2.12.1 and BA.2.75 were also constructed by adding more mutations to the BA.2 construct. To generate BA.2.75, K147E, W152R, F157L, I210V, G275S, G446S, and N460K were added to the BA.2 skeleton. 339D was also changed to 339H in BA.2 S, and 493R was reversed to 493Q in BA.2, as in the ancestral strain. To test the effect of single mutations, D339H, G446S, N460K, and R493Q were individually introduced into the BA.2 skeleton. The resulting pcDNA3.1 plasmids containing the S gene were used to generate pseudoviral particles with lentiviral packaging vectors and encoding transfer vectors.

[0375] Pseudovirus neutralization test Details of the pseudovirus neutralization test were previously described (Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16(2021):4220-4236), with some modifications. Briefly, the neutralizing activity of potent monoclonal antibodies (mAbs) generated from donors who recovered from Omicron and Beta infection, as well as donors infected in the UK during the early stages of the pandemic, was tested against Victoria, Omicron-BA.1, BA.1.1, BA.2, BA.2.11, BA.2.12.1, BA.2.13, BA.3, BA.4.6, BA.4 / 5, BA.2.75, and BA.2+N460K. Four-fold serial dilutions of each mAb were incubated with pseudovirus particles at 37°C and 5% CO2 for 1 hour. Next, stable HEK293T / 17 cells expressing human ACE2 were added to the mixture at 1.5 × 10⁴ cells / well. After transduction, the culture supernatant was removed 48 hours later, and 50 μL of 1:2 Bright-Glo™ luciferase assay system (Promega, USA) in 1 × PBS was added to each well. The reaction mixture was incubated at room temperature for 5 minutes, and firefly luciferase activity was measured using CLARIOstar® (BMG Labtech, Ortenberg, Germany). mAb neutralization rates were calculated in comparison to the control. Probit analysis was used to estimate the dilution value (PVNT50) that inhibits half of the maximum pseudotyped lentivirus infection.

[0376] To measure the neutralizing activity of convalescent plasma / serum samples or vaccine serum, 3-fold serial dilutions of the samples were incubated with pseudovirus particles for 1 hour, and the same strategy as for mAbs was applied.

[0377] DNA manipulation Cloning was performed using an unrestricted approach (Peleg and Unger, 2014). Mutagenic megaprimers were PCR amplified (KAPA HiFi HotStart ReadyMix, Roche, Switzerland, catalog number KK3605), purified using NucleoSpin® gel and PCR Clean-up kit (Nacherey-Nagel, Germany, reference number 740609.50), and cloned into pJYDC1 (Adgene ID: 162458) (Zahradnik et al., 2021a). The parental pJYDC1 molecule was cleaved by DpnI treatment (1 hour, NEB, USA, cat.R0176), and the reaction mixture was electroporated into E. coli Cloni® 10G cells (Lucigen, USA). The accuracy of mutagenesis was verified by sequencing.

[0378] Spike and RBD cloning Expression plasmids for wild-type and omicron BA.1 spike, as well as RBDs for BA.1 and BA.2, were constructed to encode human codon-optimized sequences from BA.1 (EPI_ISL_6640917) and BA.2 (EPI_ISL_6795834.2). The constructs of the wild-type and BA.1 spike and RBD plasmids are the same as those previously described (Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184.8(2021):2183-2200). Using a synthetic codon-optimized RBD fragment of BA.2 as a template, the constructs were amplified by PCR and cloned into pNEO vectors as previously described (Dejnirattisai et al., 2021a; Supasa et al., 2021; Zhou et al., 2021). The structure was confirmed by Sanger sequencing.

[0379] To generate His-tagged constructs of BA.4 / 5RBD, the BA.2RBD construct was used as a template (Nutalai et al., 2022), and site-directed PCR mutagenesis was performed to introduce the L452R, F486V, and R493Q mutations. The gene fragments were amplified using pNeoRBD333Omi|F(5'-GGTTGCGTAGCTGAAACCGGTCATCACCATCACCATCACACCAATCTGTGCCCTTTCGAC-3') and pNeoRBD333_R(5'-GTGATGGTGGTGCTTGGTACCTTATTACTTCTTGCCGCACACGGTAGC-3') and cloned into pNeo vectors (Supasa et al., 2021, “Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera”.Cell 184, 2201-2211 e2207). To generate BA.4 / 5 RBD constructs containing the BAP-His tag, gene fragments were amplified with RBD333_F(5'-GCGTAGCTGAAACCGGCACCAATCTGTGCCCTTTCGAC-3') and RBD333_BAP_R(5'-GTCATTCAGCAAGCTCTTCTTGCCGCACACGGTAGC-3') and cloned into the pOPINTTGneo-BAP vector (Huo et al., 2020, “Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2”. Nature structural & molecular biology 27, 846-854.). Cloning was performed using the ClonExpress II One Step Cloning Kit (Vazyme). Constructs were validated by Sanger sequencing after plasmid isolation using the QIAGEN Miniprep kit (QIAGEN).

[0380] To generate the BA.2.75 RBD construct, the BA.2 spike construct was used as a template (Nutalai et al., 2022), and site-directed PCR mutagenesis was performed using the primers listed in Figure 26 to introduce the D339H, G446S, N460K, and R493Q mutations. The gene fragments were amplified with D339H_pNeoF and RBD333_BAP_R (Figure 26) and cloned into the pOPINTTGneo-BAP vector (Huo et al., 2020, “Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.” Nature structural & molecular biology 27, 846-854). To generate the BA.2+R493Q RBD construct, the BA.2 spike construct was used as a template, and site-directed PCR mutagenesis was performed to introduce the R493Q mutation using the primers listed in Figure 26. The gene fragments were amplified with pNeoRBD333Omi_F and BD333_BAP_R and cloned into the pNeo vector (Supasa et al., 2021 “Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.” Cell 184, 2201-2211 e2207). Cloning was performed using the ClonExpress II One Step Cloning Kit (Vazyme). The constructs were validated by Sanger sequencing after plasmid isolation using the QIAGEN Miniprep kit (QIAGEN).

[0381] RBD production The plasmid encoding RBD was transfected into Expi293F® cells (ThermoFisher) via PEI and cultured in FreeStyle® 293 Expression Medium (ThermoFisher) at 30°C and 8% CO2 for 4 days. To express biotinylated RBD, the RBD-BAP plasmid was transfected with pDisplay-BirA-ER (Addgene plasmid 20856; encoding an ER-localized biotin ligase) in the presence of 0.8 mM D-biotin (Sigma-Aldrich).

[0382] Production of BA.2.75 RBD The plasmid encoding RBD was transfected into Expi293F® cells (ThermoFisher) via PEI and cultured in FreeStyle® 293 Expression Medium (ThermoFisher) at 37°C for 1 day, followed by 3 days of culture at 30°C in 8% CO2. To express biotinylated RBD, the RBD-BAP plasmid was transfected with pDisplay-BirA-ER (Addgene plasmid 20856; encoding an ER-localized biotin ligase) in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioning medium was diluted 1:2 in binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBD was purified by His tag coupling on a 5 mL HisTrap nickel column (GE Healthcare), followed by purification by Superdex75 10 / 300GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.

[0383] Protein synthesis Protein expression and purification were performed as previously described (Dejnirattisai et al., 2021a; Zhou et al., 2020). Briefly, plasmid-coding proteins were transiently expressed in HEK293T (ATCC CRL-11268) cells. Conditional media were enriched using a QuixStand benchtop system. His-tagged omicron RBDs were purified using a 5 mL HisTrap nickel column (GE Healthcare) and further refined using a Superdex 75 HiLoad 16 / 60 gel filtration column (GE Healthcare). Twin-strep-tagged omicron spikes were purified using Strep-Tactin XT resin (IBA lifesciences). Approximately 4 mg of ACE2 was mixed with homemade His-tagged 3C protease and DTT (final concentration 1 mM). After incubation at 4°C for 1 day, the samples were passed through a 5 mL HisTrap nickel column (GE Healthcare). His-tagged proteins were removed by nickel column, and purified ACE2 was recovered and concentrated.

[0384] IgG mAb and Fab purification To purify full-length IgG mAbs, the supernatant from mAb expression was collected, filtered using a vacuum filter system, and loaded onto protein A / G beads overnight at 4°C. The beads were washed three times with PBS, and IgG was eluted using 0.1 M glycine pH 2.7. The eluate was neutralized with Tris HCl pH 8 buffer to a final pH of 7. The IgG concentration was determined by spectrophotometric analysis and the buffer was replaced with PBS. To express and purify Fab158 and EY6A, Fab heavy and light chain expression plasmids were co-transfected into HEK293T cells by PEI. After culturing the cells in 5% CO2 at 37°C for 5 days, the culture supernatant was collected and filtered using a 0.22 mm polyethersulfone filter. Fab158 was purified using Strep-Tactin XT resin (IBA lifesciences), and Fab EY6A was purified using Ni-NTA columns (GE HealthCare) and Superdex 75 HiLoad 16 / 60 gel filtration columns (GE Healthcare). AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, and Lilly, and Adagio antibodies were provided by Adagio. For the antibodies, the heavy and light chains of the specified antibodies were transiently transfected into 293Y cells, and the antibodies were purified from the supernatant on Protein A. Using the Pierce Fab Preparation Kit (Thermo Fisher), Fab fragments of 58 and beta-55 were digested from purified IgG with papain according to the manufacturer's protocol.

[0385] Surface plasmon resonance Surface plasmon resonance experiments were performed using a Biacore T200 (GE Healthcare). All assays were performed at 25°C with HBS-EP (Cytiva) running buffer.

[0386] A protein A sensor chip (Cytiva) was used to determine the binding rate between SARS-CoV-2 RBD and ACE2 / monoclonal antibody (mAb). ACE2-Fc or mAb was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected into two flow cells at a flow rate of 30 μl / min at five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetic program. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0387] A CM5 sensor tip was used to measure the binding rate between the SARS-CoV-2 spike and ACE2. The sensor tip was first activated by injecting an equal mixture of EDC and NHS (Cytiva) at 20 μL / min for 300 seconds, followed by injecting a 20 μg / mL spike sample in 10 mM sodium acetate pH 5.0 (Cytiva) into the sensor tip's sample flow cell at 10 μL / min, and finally activating it by injecting 1.0 M ethanolamine-HCl, pH 8.5 (Cytiva) at 20 μL / min for 180 seconds. The reference flow cell was left blank. ACE2 was injected into two flow cells at a flow rate of 30 μL / min in five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetic program. Running buffer was also injected using the same program for background subtraction.

[0388] All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1. A Biotin CAPture Kit (Cytiva) was used to measure the binding rate between RBD and mAb Omi-32 / Omi-42. Biotinylated RBD was immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. mAb Fab was injected into two flow cells at a flow rate of 30 μl / min in five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetics program. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0389] To measure the binding affinity between BA.4 / 5 RBD and mAb Omi-12, a Protein A sensor tip (Cytiva) was used. Ig Omi-12 was immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. RBD was injected into two flow cells at a flow rate of 30 μl / min in seven concentration ranges prepared by stepwise 2-fold dilution. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Prism9 (GraphPad).

[0390] To compare the binding profiles of mAb Omi-06 / Omi-25 / Omi-26 to BA.2 and BA.4 / 5 RBD, a Protein A sensor chip (Cytiva) was used. IgG-formulated mAbs were immobilized at similar levels (approximately 350 RU) on the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected once into both flow cells at 200 nM and a flow rate of 30 μl / min. Running buffer was also injected using the same program for background subtraction. Sensorgrams were plotted using Prism9 (GraphPad).

[0391] To compare the binding profiles of mAb Omi-02 / Omi-23 / Omi-31 to BA.2 and BA.4 / 5 RBD, a Biotin Capture Kit (Cytiva) was used. Biotinylated BA.2 and BA.4 / 5 RBD were immobilized on the sample flow cell of the sensor tip at similar levels (approximately 120 RU). The reference flow cell was left blank. A single injection of mAb Fab was performed into two flow cells at 200 nM and a flow rate of 30 μl / min. Running buffer was also injected using the same program for background subtraction. Sensorgrams were plotted using Prism9 (GraphPad).

[0392] A protein A sensor tip (Cytiva) was used to measure the binding rate between BA.2.75 or BA.2+R493Q RBD and ACE2. ACE2-Fc was immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. RBD was injected into two flow cells at a flow rate of 30 μl / min at five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetic program. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0393] To confirm the binding rate between BA.2.75RBD and ACE2, a Biotin CAPture Kit (Cytiva) was used. Biotinylated ACE2 (bio-ACE2) was immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. BA.2.75 RBD was injected into two flow cells at a flow rate of 30 μl / min in five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetics program. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0394] A Biotin CAPture Kit (Cytiva) was used to measure the binding rate between BA.2.75 or BA.2 RBD and mAbs. Biotinylated RBDs were immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. Fabs of Omi-18 or Omi-32 were injected into two flow cells at a flow rate of 30 μl / min in five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetic program. To bind Omi-20 to bio-BA.2 RBD, Fabs of Omi-20 were injected into two flow cells at a flow rate of 30 μl / min in five concentration ranges prepared by stepwise 2-fold dilutions using a single-cycle kinetic program. To bind Omi-20 to bio-BA.2.75 RBD, Fabs of Omi-20 were injected into two flow cells at a flow rate of 30 μl / min in eight concentration ranges prepared by stepwise 2-fold dilutions. For background subtraction, the same program was used to inject a running buffer. All data were fitted to a 1:1 coupled model using Biacore T200 Evaluation Software 3.1.

[0395] To compare the binding profiles of BA.2 and BA.2.75 RBD to mAb Omi-29, a Biotin Capture Kit (Cytiva) was used. Biotinylated BA.2 and BA.2.75 RBD were immobilized on the sample flow cell of the sensor tip at similar levels (approximately 110 RU). The reference flow cell was left blank. A single injection of mAb Fab was performed into the two flow cells at 1 μM and a flow rate of 30 μl / min. Running buffer was also injected using the same program for background subtraction. Sensorgrams were plotted using Prism9 (GraphPad).

[0396] To compare the binding profiles of mAb Omi-36 to BA.2 and BA.2.75 RBD, sensor tip protein A (Cytiva) was used. IgG-formulated mAb Omi-36 was immobilized on the sample flow cell of the sensor tip. The reference flow cell was left blank. RBD was injected into both flow cells once at 200 nM and a flow rate of 30 μl / min. Running buffer was also injected using the same program for background subtraction. Sensorgrams were plotted using Prism9 (GraphPad).

[0397] IgG mAb and Fab generation AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, and Lilly; Adagio antibodies were provided by Adagio; and LY-CoV1404 was provided by LifeArc. As previously described (Nutalai et al., 2022), for in-house antibodies, the heavy and light chains of the specified antibodies were transiently transfected into 293Y or 293T cells, and the antibodies were purified from the supernatant on Protein A. Using the Pierce Fab Preparation Kit (Thermo Fisher), Fab was digested with papain from purified IgG according to the manufacturer's protocol.

[0398] Quantification and statistical analysis Statistical analysis is reported in the results and figure descriptions. Neutralization was measured by FRNT. The percentage of focus reduction was calculated, and IC50 (FRNT50) was determined using the probit program of the SPSS package. Wilcoxon's matched pair signed-rank test was used for analysis, and the two-tailed p-value was calculated based on the geometric mean.

[0399] crystallization The RBD protein was deglycosylated with endoglycosidase F1 before use for crystallization. Omicron BA.1-RBD was mixed separately with Omi-12 and Beta-54 Fab in a 1:1:1 molar ratio, with a final concentration of 7 mg / ml. These complexes were incubated separately at room temperature for 30 minutes. As previously described (Walter et al., 2003, Journal of Applied Crystallography 36, 308-314), the initial screening of crystals was set up in a Cartesian Robot Crystalquick 96-well X plate (Greiner Bio-One) using nanoliter sitting-drop vapor diffusion, with each drop containing 100 nL of protein plus 100 nL of reservoir.

[0400] Crystals of the BA.1-RBD / Omi-12 / beta-54 complex were formed under Hampton Research PEGRx conditions 1-46 containing 0.1 M sodium citrate tribase dihydrate pH 5.0 and 18% (w / v) PEG 20000. The BA.1-RBD / Omi-12 / beta-54 complex was screened with Hampton Research ammonium sulfate screen C2 containing 2.4 M (NH4)2SO4 and 0.1 M citrate pH 5.0, but under these conditions, only crystals of FabOmi-12 were formed.

[0401] BA.2.75 Crystallization of RBD Purified BA.2.75 RBD was deglycosylated with endoglycosidase H1 and mixed with ACE2 in a 1:1 molar ratio to a final concentration of 13.0 mg / ml. As previously described (Walter et al., 2003), the initial screening of crystals was set up in a Crystalquick 96-well X plate (Greiner Bio-One) using a Cartesian Robot with nanoliter sitting-drop vapor diffusion, with each drop containing 100 nL of protein plus 100 nL of reservoir. Crystals of the BA.2.75 RBD-ACE2 complex were formed under Hampton Research PEGRx conditions 2-25 containing 0.1% (w / v) n-octyl-bD-glucoside, 0.1 M sodium citrate tribase dihydrate pH 5.5, and 22% (w / v) PEG 3350. Diffraction data was collected at 100K at beamline I03 of Diamond Light Source in the UK using an automated queuing system that enables unattended automated data collection (https: / / www.diamond.ac.uk / Instruments / Mx / I03 / I03-Manual / Unattended-Data-Collections.html).

[0402] X-ray data acquisition, structural determination, and refinement. Diffraction data was collected at 100K on beamline I03 at Diamond Light Source, UK. All data was collected as part of an automated queue system enabling unattended automated data collection (https: / / www.diamond.ac.uk / Instruments / Mx / I03 / I03-Manual / Unattended-Data-Collections.html). The crystal was mounted in a loop, pre-frozen, and immersed for 1 second in an antifreeze containing 25% glycerol and 75% mother liquor. Diffraction images rotated 0.1° were recorded with an Eiger2 XE 16M detector (exposure time 0.018 seconds per image, beam size 80 × 20 μm, beam transmittance 10%, wavelength 0.9762 Å). The data were indexed, integrated, and scaled using the automated data processing program Xia2-dials (Winter, 2010, Journal of applied crystallography 43, 186-190; Winter et al., 2018, Acta Crystallogr D Struct Biol 74, 85-97). For each dataset, 360° data was collected from a single crystal.

[0403] The structure was determined by molecular substitution using PHASER (McCoy et al., 2007, J Appl Crystallogr 40, 658-674). The VhVl and ChCl domains with the highest sequence similarity to previously measured SARS-CoV-2 RBD / Fab structures (Dejnirattisai et al., 2021, Cell 184, 2183-2200 e2122; Dejnirattisai et al., 2021, Cell 184, 2939-2954 e2939; Huo et al., 2020, Cell Host Microbe 28, 445-454; Liu et al., 2021, Cell 184, 4220-4236 e4213; Supasa et al., 2021, Cell 184, 2201-2211 e2207; Zhou et al., 2021, Cell 184, 2348-2361 e2346; Zhou et (al., 2020, Nature Structural & Molecular Biology 27, 950-958) was used as the current search model for determining each structure.

[0404] For all structures, model reconstruction using COOT (Emsley et al., 2010, Biological Crystallography 66, 486-501) and refinement using Phenix (Liebschner et al., 2019, Acta Crystallogr D Struct Biol 75, 861-877) were employed. Due to low resolution, only improvements to the rigid body and group B coefficients were performed on the structure of the BA.1-RBD / O-12 / beta-54 complex.

[0405] Statistics for data collection and structural refinement are given in Tables 19 and 25. Structural comparisons were performed using SHP (Stuart et al., 1979, J Mol Biol 134, 109-142), residues forming the RBD / Fab interface were identified using PISA (Krissinel and Henrick, 2007, J Mol Biol 372, 774-797), and figures were created using PyMOL (PyMOL Molecular Graphics System, version 1.2r3pre, Schroedinger, LLC).

[0406] Example 5 antibody structure The structure of the BA.1 RBD / Fab Omi-12 / Fab beta-54 ternary complex was measured at a resolution of 5.5 Å (Table 19, Figure 6A). Although no significant competition for binding between the two Fabs was observed in BLI experiments, slight collisions were observed between them. The high-resolution structure of the Omi-12 fab not forming the complex (resolution 2.1 Å, Table 19) is modeled to represent the electron density of the complex (Figures 6B, 6C). When Fab253 is superimposed on Fab Omi-12, it is suggested that Q493R may collide with the H2 loop of Fab253, although H2 in Omi-12 adopts a slightly flattened structure. This structural change is attributed to antibody maturation via the somatic mutation V53P in the heavy chain variable region of Omi-12, which forms a stacking interaction with Y489 (Figure 6D).

[0407] Both Omi-12 and antibody 253 are derived from the germline heavy chain IGHV1-58. Interestingly, like antibody 253, the other antibodies derived from the germline heavy chain IGHV1-58 described herein, namely beta-47, beta-25, antibody 55, antibody 165, and antibody 318, also have valine (V) at position 53 of the heavy chain variable region, i.e., valine (V) at position 53 in SEQ ID NO: 262 (antibody 253), SEQ ID NO: 591 (beta-47), SEQ ID NO: 461 (beta-25), SEQ ID NO: 62 (antibody 55), SEQ ID NO: 182 (antibody 165), and SEQ ID NO: 332 (antibody 318). Position 53 in these sequences corresponds to position 58 according to IMGT numbering. Based on the data, modification of any of these antibodies by substituting valine at position 53 with proline (i.e., V53P in absolute numbering, V58P in IMGT numbering) may result in an antibody that may be effective against omicrons.

[0408] Furthermore, the antibody AZD8895 (the amino acid sequence of the heavy chain variable region is provided in SEQ ID NO: 963, and the amino acid sequence of the light chain variable region is provided in SEQ ID NO: 965) is also derived from the germline heavy chain IGHV1-58 (see, for example, Nat Microbiol 6, 1233-1244 (2021)). AZD8895 has isoleucine (I) at position 53 in the heavy chain variable region, which corresponds to position 58 according to IMGT numbering. Based on the data here, modifying the heavy chain variable region AZD8895 (SEQ ID NO: 963) by substituting the isoleucine at position 53 with proline (i.e., I53P) using absolute numbering, or by substituting it with I58P using IMGT numbering, may result in an antibody that could be effective against omicrons.

[0409] Therefore, the data indicates that modifying the VH1-58 antibody to include proline at position 53 in the heavy chain variable region (corresponding to position 58 according to IMGT numbering) is particularly effective against omicrons.

[0410] ACE2 / BA.2.75 RBD structure To elucidate the high-affinity molecular mechanism, the structure of BA.2.75RBD with ACE2 was determined by crystal structure analysis (according to the method described in Example 4). As expected, the binding mode was essentially indistinguishable from those previously observed (Figure 20A), but there was significant rearrangement outside the ACE2 footprint, including rearrangement of the flexible RBD371-375 loops and alignment of part of the C-terminal 6xHis tag. Figure 20B shows a magnified view of the binding interface compared to the ACE2 / BA.2 RBD complex. In other complexes (having either R or Q at RBD493), K31 of ACE2 tends to be disordered, but in the BA.2.75 complex it aligns properly, suggesting that K31 may form potential hydrogen bonds with the glutamine side chain, thereby increasing the affinity for ACE2. Embodiments of the present invention Embodiments of the present invention are further described in the following sections: [Section 1] An antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising at least three CDRs of antibody Omi12 or one of the 27 antibodies listed in Table 3. [Section 2] (a) CDRs of at least four, five, or all six of the antibodies listed in Table 3; (b) A heavy chain variable domain containing or consisting of an amino acid sequence having at least 80% sequence identity with the heavy chain variable domain of the antibody in Table 3; (c) A light chain variable domain containing or consisting of an amino acid sequence having at least 80% sequence identity with the light chain variable domain of the antibody in Table 3; and / or (d) Heavy chain variable domains and light chain variable domains that contain or consist of amino acid sequences that have at least 80% identity with the heavy chain variable domain and light chain domain of the antibodies in Table 3, respectively. The antibody described in item 1 above, including the antibody described in item 1 above. [Section 3] The antibodies in Table 3 are, (a) Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42; (b) Omi03 and Omi12; (c) Omi03, Omi12, Omi02, Omi39 and Omi42; (d) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28 and Omi08; or (e) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38 An antibody selected from the group consisting of items 1 or 2 above. [Section 4] The antibody described in item 1 above, comprising CDRH1, CDRH2, and CDRH3 derived from a first antibody in any one of Tables 1 to 3, and CDRL1, CDRL2, and CDRL3 derived from a second antibody in any one of Tables 1 to 3, provided that the first antibody and the second antibody are different. [Section 5] The antibody described in item 4 above, comprising a heavy chain variable domain amino acid sequence having at least 80% sequence identity with the heavy chain variable domain derived from the first antibody in any one of Tables 1 to 3, and a light chain variable domain amino acid sequence having at least 80% sequence identity with the light chain variable domain derived from the second antibody in any one of Tables 1 to 3. [Section 6] The antibody according to item 4 or 5 above, wherein the first and second antibodies are derived from the same germline heavy chain v region, and optionally, the heavy chain v region is IGHV1-69, IGHV3-53, IGHV1-58, IGHV3-66, IGHV3-30, IGHV3-33, IGHV1-18, IGHV3-9, or IGHV4-31. [Section 7] The first antibody and the second antibody are, (a) Selectively including one heavy chain variable domain and one light chain variable domain of the antibodies listed in Table 4, Omi03, Omi18, Omi29, Beta-27, antibody 150, antibody 158, antibody 175, antibody 222 and antibody 269; (b) Optionally, Omi03, Omi18, Omi29, Omi16, Omi17, Omi20, Omi27, Omi36, Beta-27, Antibody 150, Antibody 158, Antibody 175, Antibody 222, Antibody 269, Antibody 40 and Antibody 398, each containing one of the heavy chain variable domains and light chain variable domains of the antibodies listed in Table 5; (c) Selectively include Omi12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253 and Antibody 318, each containing one of the heavy chain variable domains and light chain variable domains of the antibodies listed in Table 6; (d) Optionally, beta-49, beta-50, Omi02, Omi24, Omi30, Omi31, Omi34 and Omi38, each containing one of the heavy chain variable domains and light chain variable domains of the antibodies listed in Table 7; (e) Optionally, beta-22, beta-29, antibody 159, and Omi09, each containing one of the heavy chain variable domains and light chain variable domains of the antibodies listed in Table 8; (f) Beta-20, Beta-43, Omi32, and Omi33, optionally containing one heavy chain variable domain and one light chain variable domain of the antibodies listed in Table 9; (g) Optionally, antibodies 278, beta-44, omi26, and omi41 containing one of the heavy chain variable domains and light chain variable domains of the antibodies listed in Table 10; (h) optionally, antibodies 58, Omi25, Omi35 and Omi42, which contain one heavy chain variable domain and one light chain variable domain of the antibodies listed in Table 11; or (i) Selectively include one heavy chain variable domain and one light chain variable domain of the antibodies listed in Table 12, including Beta-56 and Omi23 Both are selected from the group consisting of the above, and are antibodies according to any one of items 4 to 6 above. [Section 8] The antibodies in Tables 3-12 are, (a) Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42; (b) Omi03 and Omi12; (c) Omi03, Omi12, Omi02, Omi39 and Omi42; (d) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28 and Omi08; or (e) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38 An antibody selected from the group consisting of items 1 to 7 above, as described in any one of the items 1 to 7 above. [Section 9] For example, an antibody according to any one of items 1 to 8 above, which is a full-length antibody containing an IgG1 constant region, or an Fc region containing at least one modification that prolongs the serum half-life. [Section 10] An antibody according to any one of the above items 1 to 9, which is derived from germline heavy chains IGHV1-58 such as Omi-12, beta-47, beta-25, antibody 55, antibody 165, antibody 253, and antibody 318, and contains proline at position 53 of the heavy chain variable region. [Section 11] A combination of antibodies containing two or more antibodies described in any one of the above items 1 to 10. [Section 12] It is a combination of antibodies, (a) The antibody described in any one of items 1 to 10 above; (b) An antibody comprising at least three CDRs of antibodies in Table 1 or Table 2, for example, (i) CDRs of at least four, five, or all six antibodies listed in Table 1 or Table 2; (ii) A heavy chain variable domain containing or consisting of an amino acid sequence having at least 80% sequence identity with the heavy chain variable domain of an antibody in Table 1 or Table 2; (iii) A light chain variable domain containing or consisting of an amino acid sequence having at least 80% sequence identity with the light chain variable domain of an antibody in Table 1 or Table 2; and / or (iv) Heavy chain variable domains and light chain variable domains that contain or consist of amino acid sequences that have at least 80% identity with the heavy chain variable domain and light chain domain of the antibodies in Table 1 or Table 2, respectively. Antibodies containing A combination of antibodies containing [the specified substance]. [Section 13] A combination of antibodies as described in item 11 or 12 above, comprising two, three, or four antibodies as described in any one of items 1 to 10 above. [Section 14] One or more polynucleotides encoding an antibody as described in any one of items 1 to 10 above, one or more vectors containing the polynucleotides, or a host cell containing the vector. [Section 15] A method for producing an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising culturing the host cells described in item 14 above, and isolating the antibody from the culture. [Section 16] (a) an antibody as described in any one of items 1 to 12 above, or a combination of antibodies as described in any one of items 11 to 13 above, and (b) at least one pharmaceutically acceptable diluent or carrier. [Section 17] An antibody as described in any one of items 1 to 10 above, a combination as described in any one of items 11 to 13 above, or a pharmaceutical composition as described in item 16 above, for use in a method of treating the body of a human or animal by therapeutic means. [Section 18] An antibody as described in any one of items 1 to 10 above, a combination as described in any one of items 11 to 13 above, or a pharmaceutical composition as described in item 16 above, for use in methods of treating or preventing coronavirus infection or diseases or complications related to coronavirus inf...

Claims

1. An antibody that can bind to the spike protein of the coronavirus SARS-CoV-2, (a) Heavy chain variable domain containing the sequence of Sequence ID No. 952; and (b) Light chain variable domain containing the sequence of sequence number 954 Antibodies containing this substance.

2. The antibody according to claim 1, wherein the antibody includes an Fc region.

3. The antibody according to claim 1, wherein the antibody includes an IgG1 constant region.

4. The antibody according to claim 2, wherein the Fc region includes at least one modification that prolongs the serum half-life.

5. The antibody according to claim 4, wherein the Fc region contains the M252Y / S254T / T256E(YTE) mutation.

6. The antibody according to claim 1, wherein the antibody is a single-chain antibody, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, or scFv.

7. A polynucleotide that encodes a light chain variable domain and a heavy chain variable domain of an antibody according to any one of claims 1 to 6.

8. A vector comprising one or more polynucleotides as described in claim 7.

9. A host cell comprising one or more polynucleotides as described in claim 7.

10. A host cell comprising one or more vectors as described in claim 8.

11. A method for producing an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising culturing the host cells described in claim 9 and isolating the antibody from the culture.

12. A method for producing an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, comprising culturing the host cells described in claim 10 and isolating the antibody from the culture.

13. A pharmaceutical composition, (a) an antibody according to any one of claims 1 to 6, (b) at least one pharmaceutically acceptable diluent or carrier A pharmaceutical composition containing the following:

14. An antibody according to any one of claims 1 to 6, for use in a method of treating the body of a human or animal by therapeutic means.

15. The pharmaceutical composition according to claim 13, for use in a method of treating the body of a human or animal by therapy.

16. An antibody according to any one of claims 1 to 6, for use in a disease or complication associated with SARS-CoV-2 infection or a method for treating or preventing such disease or complication.

17. The antibody according to claim 16, wherein the associated disease or complication is COVID-19.

18. The pharmaceutical composition according to claim 13, for use in a method of treating or preventing a disease or complication associated with SARS-CoV-2 infection or a disease or complication associated therewith.

19. The pharmaceutical composition according to claim 18, wherein the associated disease or complication is COVID-19.

20. The antibody according to claim 16, wherein the SARS-CoV-2 infection is caused by an alpha, beta, gamma, delta, or omicron lineage SARS-CoV-2 strain.

21. The antibody according to claim 20, wherein the Omicron strain is Omicron BA. 2.11, Omicron BA. 2.12.1, Omicron BA. 2.13, Omicron BA. 2.3.20, Omicron BA. 2.10.4, Omicron BA. 1, Omicron BA. 1.1, Omicron BA. 2, Omicron BA. 2.75, BA. 2.75.2, Omicron BA. 3, Omicron BA. 4.6, Omicron BA. 4 / 5, Omicron BJ. 1, Omicron BS. 1, Omicron BN. 1, Omicron BF. 7, Omicron BQ. 1, Omicron BQ. 1.1, Omicron XBB and / or Omicron XBB.

1.

22. The pharmaceutical composition according to claim 18, wherein the SARS-CoV-2 infection is caused by an alpha, beta, gamma, delta, or omicron strain of SARS-CoV-2.

23. The pharmaceutical composition according to claim 22, wherein the Omicron strain is Omicron BA. 2.11, Omicron BA. 2.12.1, Omicron BA. 2.13, Omicron BA. 2.3.20, Omicron BA. 2.10.4, Omicron BA. 1, Omicron BA. 1.1, Omicron BA. 2, Omicron BA. 2.75, BA. 2.75.2, Omicron BA. 3, Omicron BA. 4.6, Omicron BA. 4 / 5, Omicron BJ. 1, Omicron BS. 1, Omicron BN. 1, Omicron BF. 7, Omicron BQ. 1, Omicron BQ. 1.1, Omicron XBB and / or Omicron XBB.

1.

24. An in vitro method for identifying the presence of SARS-CoV-2 in a sample, comprising contacting the sample with an antibody according to any one of claims 1 to 6, and detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates the presence of SARS-CoV-2 in the sample.

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