Antibody capable of binding to spike protein of coronavirus SARS-COV-2

Abstract

The present disclosure relates to antibodies useful in the prevention, treatment and / or diagnosis of coronavirus infections, as well as diseases and / or complications associated with coronavirus infections, including COVID-19. In particular, the present disclosure relates to antibodies capable of binding to the spike protein of the coronavirus SARS-CoV-2 and uses thereof.

Description

Technical Field

[0001] The present invention relates to a method for preventing, treating and / or diagnosing coronavirus infection and

[0002] Antibodies to illness and / or complications associated with coronavirus infection, including COVID-19. Background Art

[0003] In December 2019, the first case of a severe viral acute respiratory syndrome named COVID-19 was reported. The virus spread rapidly around the world, causing a pandemic with more than 200 million confirmed infections and more than 4.4 million deaths in 12 months. The pathogen, SARS-CoV-2, is a betacoronavirus related to SARS-CoV-1 and MERS coronaviruses, both of which cause severe respiratory syndromes.

[0004] In the months since SARS-CoV-2 was identified as the pathogen of COVID-19, our understanding of the disease and the virus has advanced dramatically. There are now a number of validated treatments, including dexamethasone and tocilizumab, as well as monoclonal antibodies (mAbs), which have been shown to be effective when used in both preventive and therapeutic settings (Baum et al., 2020, Science 369, 1014-1018). Despite these advances, the pandemic remains far from under control, resulting in successive waves of infection.

[0005] Coronaviruses have four structural proteins: nucleocapsid, envelope, membrane, and spike (S) protein. The spike protein is the most important surface protein. It has an elongated trimeric structure and is responsible for engaging the target cell and triggering fusion of the virus with the host membrane. The spike proteins from both SARS-CoV-2 and SARS-CoV-1 use angiotensin-converting enzyme 2 (ACE2) as a cell surface receptor. ACE2 is expressed in many tissues, including epithelial cells of the upper and lower respiratory tracts.

[0006] The S protein consists of two subunits, S1, which mediates receptor binding, and S2, which is responsible for the fusion of the viral and host cell membranes. It is a dynamic structure that can transition to a post-fusion state after receptor binding or trypsin treatment by cleavage between S1 and S2. In some SARS-CoV-2 sequences, a furin cleavage site is inserted between the S1 and S2 subunits, and mutations in the cleavage site attenuate disease in animal models. The S1 fragment occupies the membrane-distal apex of S and can be subdivided into an N-terminal domain (NTD) and a receptor binding domain (RBD). While both regions are immunogenic, the RBD contains an interaction surface for ACE2 binding. Although the RBD is usually squeezed down against the top of S2, it can swing up to engage ACE2. Monoclonal antibodies (mAbs) recognize one or both of the "up" and "down" conformations.

[0007] The S protein is relatively conserved between SARS-CoV-2 and SARS-CoV-1 (76%), but the RBD and NTD are less conserved (74% and 50%, respectively) than the S2 domain (90%). Conservation against MERS-CoV and seasonal human coronaviruses is much lower (19-21%). Overall, SARS-CoV-2 antibodies show limited cross-reactivity, even with SARS-CoV-1.

[0008] S is involved in the attachment of the virus to target cells via the interaction of cell surface expressed ACE2 with the S receptor binding motif (also known as the ACE-2 footprint), a 25 amino acid patch located at the tip of the receptor binding domain (RBD) in the S1 fragment of the spike protein. After attachment, cleavage of S releases S1, allowing a major conformational change in S2 to expose the hydrophobic fusion loop to perform fusion of the viral and host cell membranes, thereby releasing the viral genome into the host cell cytoplasm to initiate viral replication. Analysis of a large number of mAbs generated from individuals infected with SARS-CoV-2 showed that mAbs bind to multiple epitopes on S1 and S2. Most mAbs generated against the original strain of SARS-CoV-2, while able to bind S with high affinity, show little or no neutralizing activity. Genomic surveillance of SARS-CoV-2 has identified thousands of mutations in structural and non-structural proteins. However, at the end of 2020, viral variants were described as rapidly becoming the dominant strain locally and causing global spread, and were designated as variants of concern (VoC).

[0009] Alpha (B.1.1.7) was first identified in the United Kingdom and has increased transmission rates. B.1.1.7 has 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. Beta (501Y.V2, also known as B.1.351) was first reported in South Africa. Gamma (P.1, 501Y.V2) were first reported in Brazil and have 10 and 12 amino acid changes in the spike protein, respectively. Delta was first reported in India and has now spread globally, causing outbreaks in multiple countries. Omicron BA.1 was first reported in southern Africa in late November 2021 and spread around the world, becoming the dominant variant in many countries, almost completely replacing Delta.

[0010] A series of sublineages of Omicron have emerged, including BA.1.1, BA.2, BA.2.12.1, BA.2.75 and BA.4 / 5, which have surpassed previous strains and become regionally or globally dominant. More than 30 mutations were found in the Omicron S protein, including 15 substitutions in the RBD, resulting in increased transmissibility (Suzuki et al., 2022 “Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.” Nature 603, 700-705) and widespread and substantial reductions in neutralizing antibody titers (Dejnirattisai et al., 2022 “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell 185, 467-484e415).

[0011] Omicron BA.2 was reported at about the same time as BA.1. The proportion of Omicron infections caused by BA.2 has been increasing in several countries and has become the dominant sublineage in Denmark and India.

[0012] BA.1.1 (containing an additional R346K mutation in the RBD) once accounted for about 40% of Omicron sequences worldwide and about 35%-60% in the UK and the US (Iketani et al., 2022 “Antibody evasion properties of SARS-CoV-2 Omicron sublineages.” Nature 604, 553-556), but was quickly overtaken by BA.2. As of August 2022, BA.2 (containing 8 unique substitutions in S, including 6 within the RBD, and lacking the 13 mutations found in BA.1 (Nutalai et al., 2022)) has become the dominant strain worldwide. Recently, BA.2.12.1 has been identified in multiple countries and has caused large regional outbreaks in North America (accounting for 58% of sequences as of May 25, 2022) (Del Rio and Malani, 2022, “COVID-19 in 2022—The Beginning of the End or the End of the Beginning?” JAMA 327, 2389–2390).

[0013] It is now becoming clear that BA.2 has a small transmission advantage over BA.1, although there is no evidence of increased disease severity. In early April 2022, two new Omicron lineages were reported in Gauteng Province, South Africa, named BA.4 and BA.5. BA.4 and BA.5 (with identical S sequences) became the dominant Omicron strains in Gauteng Province, triggering a new wave of infections in South Africa.

[0014] Since June 2022, BA.4 / 5 (with higher receptor binding affinity and significantly enhanced escape from antibody responses (Tuekprakhon et al., 2022 “Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5from vaccine and BA.1 serum.” Cell 185, 2422-2433e2413)) has rapidly spread from South Africa to the world and has now become the new dominant strain in the world, with BA.5 dominating in many regions. These variants (especially BA.5) now account for the majority of sequenced cases in many countries.

[0015] In early May 2022, a new Omicron sublineage emerged in India, named BA.2.75. This strain has since spread to many countries, including the United Kingdom, the United States, Australia, Germany, and Canada. However, the true prevalence of BA.2.75 is difficult to determine because sequencing in many countries is sporadic and has been greatly reduced.

[0016] All of these variants contain multiple mutations in S and include changes in the RBD, NTD, and in some cases 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 in or immediately adjacent to the ACE2 interaction surface, where they have the potential to modulate ACE2 interactions and disrupt the binding of neutralizing antibodies. The increased affinity for ACE2 interactions was primarily for Alpha, Beta, Gamma, and Delta (7, 19, 19, 2-fold, respectively), and may play a role in increasing viral transmissibility. Omicron contains an unprecedented number of mutations, concentrated in the spike (S) gene, which carries 30 substitutions, plus a deletion of 6 residues and an insertion of 3 residues. Omicron BA.1 (RBD mutations G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H) contains unique mutations S371L, G446S and G496S, and in some isolates contains R346K (BA.1.1), while BA.2 carries S371F, T376A, D405N and R408S. BA.3 does not contain unique mutations relative to BA.1 and BA.2 and appears to be a fusion of the two, being BA.1-like at the N-terminus and converted to BA.2-like at the C-terminus by mutation G496S.

[0017] Compared with BA.2, BA.2.75 contains multiple mutations in the S protein, including four substitutions in the NTD (W152R, F157L, I210V and G257S) and four substitutions in the RBD: D339H, G446S, N460K and R493Q.

[0018] Three new variants related to BA.2 have also been detected in multiple countries, namely BA.2.11, BA.2.12.1 and BA.2.13. Compared with the BA.2 spike protein receptor binding domain (RBD), these new variants contain single mutations L452R, L452Q and L452M ( Fig.29). Among them, BA.2.12.1, first identified in New York, dominates in the United States, accounting for approximately 58% of SARS-CoV-2 isolates as of May 25, 2022. While L452R is found in the Delta and Kappa variants, and L452Q is found in the Lambda variant, L452M is new.

[0019] BA.2.13 is expected to be a relatively modest change given the physicochemical properties of the side chain at residue 452; the L to M change would increase the size of the side chain, but it would still be hydrophobic. The L to Q change in BA.2.12.1 would introduce some polar character, while BA.2.11 would be the most radical, with the L to R change introducing a large basic amino acid.

[0020] Additional variants (i.e., BA.4 and BA.5, which have the same S sequence) appear to have evolved from BA.2. The sequences of BA.4 and BA.5 are highly related to that of BA.2, but contain additional mutations. Specifically, residues 69 and 70 of the NTD have been deleted (also found in Alpha, BA.1, and BA.3) and they contain two additional substitutions in 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 as in the Victoria / Wuhan strain. When looking at the RBD, BA.4 and BA.5 have assembled mutations at all previously described positions in VoC Alpha (N501Y), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y), Delta (L452, T478K), with the only difference being E484A instead of E484K Beta and Gamma in BA.4 and BA.5.

[0021] As of September 2022, a new variant related to BA.4 / 5 (designated BA.4.6) has emerged and expanded in the United States, where BA.5 is dominant (87.5% prevalence as of September 10, 2022, increasing from less than 2% of sequences in early July 2022 to more than 6% in mid-August 2022). Compared to BA.4 / 5, BA.4.6 contains two additional mutations in the spike protein (S), R346T in the RBD and N658S in the C-terminal domain. The R346T mutation has raised concerns about enhancing antibody evasion of BA.4 / 5, as the R346K mutation in BA.1.1 reduces serum neutralization and weakens the activity of many monoclonal antibodies (mAbs) compared to BA.1 (Nutalai et al., 2022). SARS-CoV-2 detection kits using monoclonal antibodies have also been developed. Examples include lateral flow tests such as Innova (SARS-CoV-2 Antigen Rapid Qualitative Test) and Quidel (Sofia 2 SARS Antigen FIA). However, these tests are reported to be highly inaccurate.

[0022] As of January 2023, additional variants have emerged, such as BQ.1 and XBB, which carry up to 8 additional RBD amino acid substitutions compared to BA.2.

[0023] The structure-function profile of a panel of monoclonal antibodies (mAbs) isolated from infected cases has provided considerable insight into the antigenicity of S and the mechanisms of neutralization. Most potent neutralizing antibodies bind at or very close to the footprint of ACE2 and act by blocking ACE2 interactions, thereby preventing cell attachment and infection. The second interaction site of the potent mAbs is close to the N-linked glycan at position N343, taking S309 as an example, these antibodies do not block ACE2 interactions but act to destabilize the S trimer. A third group of potent mAbs binds to the N-terminal domain in S1 and their mode of action is currently unknown. Another RBD epitope of potential interest is located outside the ACE2 footprint, and although the mAbs binding here are not potent neutralizers, they can effectively protect in vivo (Huo et al., 2020; Sun et al., 2021; Yuan et al., 2020; Zhou et al., 2020).

[0024] After BA.5, several new trends were observed in the evolution of Omicron: i) the emergence of "second-generation" BA.2 variants (including derivatives of BA.5) - variants with long phylogenetic branch lengths, multiple antigenic mutations, and lack of genetic intermediates, such as BA.2.75, BJ.1, BS.1, BA.2.10.4, and BA.2.3.20 (van der Straten et al., 2022. Immunity 55, 1725-1731); and ii) accelerated antigenic drift, which can be seen in BA.5 (Tuekprakhon et al., 2022) and in these second-generation BA.2 lineages, especially BQ.1 and BA.2.75 (https: / / nextstrain.org / nextclade / sars-cov-2 / 21L). Finally, recombination between two of these second-generation variants (BJ.1 and BM.1.1.1) gave rise to XBB. Many of these variants show a large degree of convergent evolution in known antigenic RBD residues, and mutations are located in regions that may threaten neutralizing antibody binding, leading to further escape from protection from infection provided by vaccines or previous SARS-CoV-2 infection, including previous Omicron infection.

[0025] Currently, many lineages within the BA.2 and BA.5 branches are growing rapidly. Most striking is the large degree of convergent evolution, especially at antigenic 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 the back mutation R493Q), such as BA.4.6 and BF.7 (R346T), BA.4.7 (R346S), BQ.1 (K444T, N460K) and BQ.1.1 (R346T, K444T, N460K); and examples from the BA.2.75 branch (naturally containing G446S, N460K and the back mutation R493Q), BA.2.75.2 (R346T and F486V), BN.1 (R346T, K356T, F490S). There are several other examples of second generation BA.2 variants, such as BJ.1 (also known as BA.2.10.1.1; R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (G446S, F486P, S494P and R493Q reversion mutations), BS.1 (BA.2.3.2.1; R346T, L452R, N460K, G476S), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and Q493R reversion mutations) and finally BA.2.75x BJ.1 recombinant XBB (which contains R346T, L368I, V445P, G446S, N460K, F486S, F490S relative to BA.2).

[0026] These second-generation BA.2 variants have become globally dominant, with BQ.1 alone accounting for 50% of infections as of December 27, 2022 ( https: / / cov-spectrum.org / explore / World / AllSamples / Past6M). And XBB.1.5 (XBB.1+F486P) is expanding rapidly in North America.

[0027] Outside of the RBD, the extent of convergent evolution is less but still present. Many second-generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in the VoC, such as Δ~144 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).

[0028] All currently approved SARS-CoV-2 vaccines are designed to induce antibody (and T cell) responses against S and contain S sequences found in the original ancestral strain. Therefore, there is particular interest in whether S mutations in VoC could cause immune escape, leading to vaccine failure or susceptibility to reinfection in previously infected individuals.

[0029] The extensive mutation burden in Omicron S abolishes the activity of most mAbs binding to the three potent antibody binding sites mentioned above (ACE-2 footprint, N343 glycan periphery, and NTD). This results in a severe reduction or complete loss of serum neutralization capacity due to natural infection or vaccination, leading to the enhanced transmissibility and explosive spread of Omicron.

[0030] The object of the present invention is to identify additional improved antibodies that can be used to prevent, treat and / or diagnose coronavirus infections and diseases and / or complications associated with coronavirus infections, including COVID-19, in particular Omicron variants of concern (VoC) and yet to be identified variants with additional mutations in the ACE-2 footprint, RBD and / or NTD in the spike protein of SARS-CoV-2. Summary of the invention

[0031] The inventors identified 28 human monoclonal antibodies (mAbs) that recognize the spike protein of SARS-CoV-2 (see Table 3). These antibodies showed potent neutralizing activity against SARS-CoV-2. Some antibodies in Table 3 showed potent neutralization against hCoV-19 / Wuhan / WIV04 / 2019 strains and antibodies from different lineages such as Victoria (Wuhan+S247R), Alpha, Beta, Gamma, Delta, Omicron (including Omicron BA.2.11, Omicron BA.2.12.1, Omicron BA.2.13, Omicron 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 SARS-CoV-2 strains (XBB and / or OmicronXBB.1 strains) are widely effective.

[0032] Many of the mAbs in Table 3 use common V genes (V genes shared by most people). The inventors have previously shown that additional antibodies can be generated by swapping the light and heavy chains of antibodies derived from the same common V genes in Tables 1, 2, and 3. Antibodies derived from the same common V genes provide particularly useful mixed-chain antibodies.

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

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

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

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

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

[0038] The present invention also provides one or more polynucleotides encoding the antibodies of the present invention, one or more vectors comprising the polynucleotides, or a host cell comprising the vector.

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

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

[0041] The present invention also provides an antibody, antibody combination or pharmaceutical composition of the present invention for use in a method of treating the human or animal body by therapy.

[0042] The present invention also provides an antibody, antibody combination or pharmaceutical composition of the present invention for use in a method for treating or preventing coronavirus infection or a disease or complication associated with coronavirus infection.

[0043] The present invention also provides a method for treating or preventing coronavirus infection or a disease or complication associated with coronavirus infection, the method comprising administering a therapeutically effective amount of an antibody, an antibody combination, or a drug combination of the present invention to the subject.

[0044] The present invention also provides a method for identifying the presence of a coronavirus or a protein fragment thereof in a sample, the method comprising (i) contacting the sample with an antibody or antibody combination 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 a coronavirus or a fragment thereof in the sample.

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

[0046] The present invention also provides the use of the antibody, antibody combination or pharmaceutical composition of the present invention for preventing, treating and / or diagnosing coronavirus infection or diseases or complications related thereto.

[0047] The present invention also provides the use of the antibody, antibody combination or pharmaceutical composition of the present invention for the manufacture of a medicament for treating or preventing coronavirus infection or diseases or complications associated therewith. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 .The BA.2 sublineage of Omicron and the generation of a panel of Omicron mAbs. Figure 1Only the first 22 Omicron antibodies disclosed in Tables 13 and 14 (i.e., Omi02 to Omi35) are involved. (A) FRNT50 titers against Victoria and Omicron BA.1 from donors used to generate Omicron mAbs are shown. (B) FACS plot showing sorting of B cells using full-length Omicron S. (C) Ratio of RBD and NTD binding antibodies found in Omicron mAbs compared to early pandemic mAbs. (D) Heavy and light chain variable gene usage. (E) Somatic mutations found in potent Omicron mAbs (FRNT50 < 100 ng / ml) compared to early pandemic group.

[0049] 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 being developed for commercial use.

[0050] Figure 3 .Neutralization of Victoria, BA.1, BA.1.1, BA.2 and BA.3 pseudoviruses. Neutralization of Victoria, BA.1, BA.1.1, BA.2 and BA.3 pseudoviruses 28 days after the second and third doses of (A) AZD1222 (n=41), (B) BNT162b2 (n=20). (C) Live virus neutralization assays were performed on Victoria, Alpha, Beta, Gamma, Delta and Omicron viruses using serum obtained <14 days and >21 days after symptom onset. (D) Neutralization of Victoria, BA.1, BA.1.1, BA.2 and BA.3 pseudoviruses by early and late sera. Geometric mean titers are shown above each column. Analyses were performed using the Wilcoxon paired signed rank test (A and B) and the Mann-Whitney test (C and D), and two-tailed P values ​​were calculated.

[0051] Figure 4. Pseudovirus neutralization curves. Pseudovirus neutralization curves of BA.1, BA.1.1, BA.2 and BA.3 against the early pandemic mAb (B) Beta mAb.

[0052] Figure 5 .and Figure 3Neutralization titers of the indicated viruses. (A) Live virus (B) Pseudovirus. Geometric mean titers are shown above each column. Wilcoxon paired signed rank test was used for analysis and two-tailed P values ​​were calculated. (C) Pseudovirus neutralization curves for selected VH1-58 mAbs and control VH3-53 mAb 222 against Victoria and Iota (S477N).

[0053] Figure 6 .Structure of BA.1 RBD with Omi-12 Fab. (A) Comparison of two ternary complexes of Omi-12 and Beta-54 Fab with BA.1 in the crystal asymmetric unit (generated by fitting the high-resolution structure of BA.1 RBD, Omi-12, and Beta-54 to the lower resolution ternary complex density) by superimposing the RBDs. The Fab in one complex is light (cartoon depiction HC red, LC blue), while the other is light. (B) Binding mode of Omi-12. (C) Close-up of the difference in binding of Omi-12 with Fab 253 to the early pandemic RBD (light blue) and Beta-47 with Beta RBD (light cyan). (D) The somatic mutation V53P facilitates refolding of the H3 loop, allowing Q493R to be accommodated in Omi-12.

[0054] Figure 7 .Pseudovirus neutralization assay of BA.4 / 5 by vaccine and BA.1 immune sera. IC50 values ​​of the specified viruses obtained using sera obtained from vaccine recipients 28 days after the third dose of vaccine (A) AstraZeneca AZD AZD1222 (n=41), (B) 4 weeks after the third dose of Pfizer BNT162b2 (n=20). Sera from volunteers suffering from breakthrough BA.1 infection were collected at (C) early symptom onset ≤114 days (median 13 days) (n=12), (D) late symptom onset ≥21 days (median 38 days) (n=16). Neutralization titers were compared with Victoria (early pandemic strain), BA.1, BA.1.1, BA.2 and BA.3. Geometric mean titers are shown above each column. Analyzed using Wilcoxon paired signed rank test, and two-tailed P values ​​were calculated.

[0055] Figure 8 .Pseudovirus neutralization assays against Omicron and commercial mAbs. Neutralization curves for a panel of 28 mAbs made from samples collected from vaccine recipients infected with BA.1. Titration curves for BA.1 were compared to BA.1, BA.1.1, BA.2, and BA.3. mAbs proposed to be affected by L452R and F486L are indicated.

[0056] Fig. 9 .Omicron sublineages compared to BA.4 / 5. (A) Comparison of S protein mutations of Omicron BA.1, BA.1.1, BA.2, BA.3, and BA.4 / 5, indicating the NTD and RBD boundaries. (B) Location of RBD mutations (grey surface, dark green for ACE2 footprint). Mutations common to all Omicron lineages are shown in white (Q493R, which is backmutated in BA.4 / 5, is shown with a cross), mutations common to BA.1 and BA.1.1 are shown in cyan, mutations unique to BA.1.1 are shown in blue, and mutations unique to BA.2 are shown in magenta. Residue 371 (yellow) is mutated in all Omicron viruses, but differs between BA.1 and BA.2. The N343 glycan is shown as a stick with a clear surface.

[0057] Fig.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 / 5RBD is severely reduced compared to that of BA.2, so binding cannot be accurately determined, as shown by a single injection of 200nM RBD over a sample flow cell containing IgG Omi-31. (BC; EI) Sensorgrams (red: raw binding curve; black: fitted curve) showing the interaction between BA.2 or BA.4 / 5RBD and selected mAbs, with kinetic data shown. (D) Affinity of BA.4 / 5RBD for Omi-12 was determined using a 1:1 binding equilibrium analysis.

[0058] Fig.11 Interactions between mAbs and BA.4 / 5 mutation sites. Overall structures (left) 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 (PDB7ZR7), (E) Wuhan-RBD / AZD8895 (PDB 7L7D), and (F) BA.1-RBD / Omi-3 (PDB 7ZF3) complexes and their interactions with BA.4 / 5 mutation sites. (Right). In the left figure, the RBD is shown as a surface representation with the BA.4 / 5 mutation site highlighted in magenta and the other two mutation sites of BA.4 / 5 at 452 and 486 shown in cyan, the Fab LC shown in blue and the HC shown as red strips. In the right figure, the side chains of the RBD, FabHC and LC are drawn as gray, red and blue sticks, respectively. In (B), L452R (green sticks) is modeled to show the possible formation of a salt bridge to D99 of CDR-H3 (yellow broken sticks). (D) The Beta-RBD / Omi-42 complex shows that the Fab does not contact any of the two BA.4 / 5 mutation sites.

[0059] Fig.12 .ACE2 RBD affinity. (A)-(D) SPR sensorgrams showing ACE2 binding to BA.4 / 5RBD (A) compared to binding to ancestral (B), BA.1 (C), and BA.2RBD (D). Data for ancestral, BA.1, and BA.2 have been reported previously (Nutalai et al., 2022). (E)-(G) Electrostatic surfaces: (E) from left to right, early pandemic, Delta, and BA.1RBD, (F) open view of BA.2RBD and ACE2 of the BA.2RBD / ACE2 complex (PDB 7ZF7), and (G) BA.4 / 5RBD (modeled based on the structure of BA.2RBD). Diamonds on ACE2 and RBD show the interaction regions.

[0060] Fig.13 .Antigenic profile. (A) Neutralization data and model (log titer values) used to calculate the antigenic profile in (B). Columns represent sera collected from vaccinated volunteers or infected patients. Rows are challenge strains: Victoria, Alpha, Delta, Beta, Gamma, BA.1, BA1.1, BA.2, BA.3 and BA.4 / 5, in order. Values ​​are colored according to deviation from a reference value; the reference value is calculated based on the serum type as the average of the neutralizing titers of the row giving the highest value. (B) Orthogonal view of the antigenic profile, showing BA.4 / 5 in the context of the previous VoC and the positions of BA.1, BA.1.1, BA.1 and BA.2 (calculated based on pseudovirus neutralization data). The distance between two positions is proportional to the reduction in neutralization titer when one of the corresponding strains is challenged with a serum obtained from infection with the other strain. Figure 6 .ACE2 / RBD affinity and antigenic profile

[0061] Fig.14Neutralization curves of VH1-58 mAb. Neutralization curves of early pandemic mAbs 253 (Dejnirattisai et al., 2021a) and Beta-47 (Liu et al., 2021b) against pseudovirus constructs of Victoria and the Omicron lineage of this group.

[0062] Fig.15 Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs. (AF) Sensorgrams (red: original binding curve; black: fitted curve) show the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs, with kinetic data shown. (GK) Binding of BA.4 / 5 RBD is severely reduced compared to that of BA.2, so binding cannot be accurately determined, as shown by a single injection of 200 nM RBD over a sample flow cell containing the indicated mAbs.

[0063] Fig.16 . Sequence changes of BA.2.75 compared to other Omicron sublineages. (A) Sequence alignment of BA.2.75 with Omicron sublineages Omicron BA.1, BA.1.1, BA.2, BA.3, and BA.4 / 5. The boundaries of the NTD and RBD are marked. (B) Surface representation of the mutated residues in the BA.2.75 RBD compared to the BA.2 RBD. The locations of the BA.2 RBD mutations are shown (grey surface with dark green ACE2 footprints), and the residues mutated in BA.2.75 are shown and marked in orange.

[0064] Fig.17 . Pseudovirus neutralization assay of vaccine and BA.1 and BA.2 immune sera against BA.2.75. IC50 values ​​of the specified viruses obtained using serum obtained from vaccine recipients 28 days after the third dose of vaccine. (A) Pfizer BNT162b2 (n=22). (B) AstraZeneca AZD AZD1222 (n=41). (C, D) Serum of volunteers infected with vaccine breakthrough BA.1 (n=16) or BA.2 (n=23). (EC) IC50 values ​​of single RBD point mutations inserted into BA.2 pseudoviruses using Pfizer BNT162b2 serum (n=22). Geometric mean titers are shown above each column. Analyzed using Wilcoxon paired signed rank test, and two-tailed P values ​​calculated.

[0065] Fig.18.ACE2 / RBD affinity. SPR sensorgrams show ACE2 binding to BA.2.75RBD using ACE2-Fc (A) or biotinylated ACE2 as ligand (B) compared to binding to BA.2 (C), BA.4 / 5 (D), Alpha (E), and BA.2+R493Q (F) RBD. Data for BA.2, BA.4 / 5, and Alpha have been previously reported in Nutalai et al., 2022, Tuekprakhon et al., 2022, and Dejnirattisai et al., 2022, respectively.

[0066] Fig.19 .Pseudovirus neutralization assays for monoclonal antibodies. (A) Neutralization curves for a panel of 28 mAbs made from samples collected from vaccine recipients infected with BA.1. Titration curves for BA.2.75 were compared with Victoria, BA.1, BA.1.1, BA.2, and BA.4 / 5. IC50 titers are shown in Table 22. (B) Pseudovirus neutralization assays for 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 are used for comparison and are taken from Tuekprakhon et al., 2022.

[0067] Fig. 20 .Structure of the BA.2.75RBD / ACE2 complex. (A) Overall structure of the BA.2.75RBD / ACE2 complex. ACE2 is shown as a green ribbon and the RBD is shown as a surface, with mutations common to BA.2 highlighted in magenta and mutations that differ highlighted in orange. (B) The interface of BA.2.75RBD (grey) and ACE2 (green) is compared to the interface of BA.2 and ACE2 (both light orange). Close-ups show the interaction of Q496R and Q493 (R493 in BA.2) with ACE2.

[0068] Fig.21Interactions between mAbs and BA.75 mutation sites. (A) Front-to-back views of the binding modes of Omi-3 (PDB, 7ZF3) and Omi-18 (PDB, 7ZFC) in complex with the omicron BA.1 RBD by overlapping RBDs. The RBD is shown as a gray surface, with mutations common to BA.2 and BA.2.75 colored in magenta, and four mutations that differ between the two colored in cyan. VH and VL are shown as ribbons and colored in red and blue for Omi-3, and light blue and light orange for Omi-18, 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 gray, the RBD associated with Omi-18 is cyan, and the color of the Fab is the same as in (A). (D) AZD1061 binds to the ancestral SARS-CoV-2 RBD (PDB, 7L7E) and (E) contacts between G446 of the RBD and CDR-L2 of the Fab. (E) AZD8895 binds to the ancestral SARS-CoV-2 spike protein RBD (PDB, 7L7E) and (F) contacts between Q493 of the RBD and CDR-H2 of the Fab. In (D)-(F), the RBD is drawn and colored the same as in (A), with HC in red and LC in blue.

[0069] Fig. 22 .Antigenic map. (A) Orthogonal view of the antigenic map showing BA.2.75 in the context of the previous VoC and the positions of BA.1, BA.1.1, BA.1 and BA.2 (calculated from pseudovirus neutralization data). The distance between two positions is proportional to the reduction in neutralization titer of one of the corresponding strains when challenged with sera obtained from infection with the other strain. No scale is provided because the figure is a projection of the three-dimensional distribution, but the change can be calculated by comparing: (i) BA.1 to BA.2, a 2.93-fold reduction, and (ii) BA.2 to BA.4 / 5, a 3.03-fold reduction. (B) Same as (A), but only the Omicron sublineage and early pandemic viruses are included to allow a more accurate projection of this subset into three-dimensional space. Note that the responses of these viruses to all sera are included in the calculations.

[0070] Fig.23 Pseudovirus neutralization assays against monoclonal antibodies. (A) Neutralization curves for a panel of 28 monoclonal antibodies made from samples collected from vaccine recipients infected with BA.1. Titration curves for BA.2.75, a single mutation in the BA.2 backbone, were compared to BA.2 and BA.2.75. IC50 titers are shown in Table 24.

[0071] Fig.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 severely reduced compared to binding of BA.2, as shown by a single injection of 1 μM Omi-29 Fab on 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 severely reduced compared to binding of BA.2, as shown by a single injection of 0.2 μM BA.2 or BA.2.75 RBD on a sample flow cell containing IgG form of Omi-36. (CH) Sensorgrams (red: original binding curve; black: fitted curve) showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs, with kinetic data shown.

[0072] Fig.25 Neutralization of BA.2.75 by multiple sets of convalescent sera collected from infection with historical variants. Neutralization titers of indicated sera against BA.2.75 and indicated pseudoviruses. Data except for BA.2.75 are taken from Tuekprakhon et al., 2022.

[0073] Fig.26 Primers used for site-directed PCR mutagenesis of BA.2.75RBD. Site-directed PCR mutagenesis was performed using the BA.2 spike construct as template. D339H, G446S, N460K and R493Q mutations were introduced using the indicated primers.

[0074] Fig. 27.BA.2.11, BA.2.12.1, and BA.2.13 were characterized by pseudovirus neutralization assays, surface plasmon resonance, and structural analysis. (a), (b) IC50 values ​​for the indicated viruses using sera obtained 4 weeks after the third dose of the following vaccines: (a) AstraZeneca AZD1222 (n=41), (b) Pfizer BNT162b2 (n=18). (c) Neutralization titers of sera from vaccinated volunteers who suffered breakthrough BA.1 infection. Comparison with neutralization titers for 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. Analysis was performed using the Wilcoxon paired signed rank test, and two-tailed p values ​​were calculated. (dg) SPR sensorgrams (red: experimental binding curve; black: fitted curve) showing ACE2 binding to RBDs of BA.2.11 (e), BA.2.12.1 (f), BA.2.13 (g) compared to binding to BA.2RBD (h), with kinetic data shown. Data for BA.2RBD are reported in Nutalai et al. (2022). (hm) Crystal structure of the BA.2.12.1RBD / Beta-27 / NbC1 complex. (h) The overall structure is shown with Cα traces of RBD (grey), Beta-27 HC (red), and LC (blue) and NbC1 (yellow). Cα of residues L452Q, F486, and Q493R (L, F, and R in BA.2, R, V, and Q in BA.4 / 5) are shown as spheres. (i) Comparison of the Beta-27 binding mode in the BA.2.12.1RBD / Beta-27 / NbC1 (RBD is surface represented, HC is red, and LC is blue), BA.4 / 5RBD / Beta-27 / NbC1 (cyan, PDB 7ZXU), and Beta RBD / Beta-27 (green, PDB 7PS1) complexes formed by overlapping RBDs. In addition to the flexible N-terminal and C-terminal regions of the RBD, the N-terminus and CDR-H1 of the Fab HC, the α2 helix, the 371-375 loop, and the G446 loop of the RBD show significant differences. CDR-L3 has a dual conformation in the BA.4 / 5RBD complex and a single conformation in the other two complexes (i). The HC N-terminus and the CDR-H1 at residue 486 contacting the RBD are different from those in the Beta and BA.4 / 5RBD complexes, the latter containing the F486V mutation. These differences are likely caused by contacts of the symmetry-related C1 nanobody, shown as grey bonds in (j). (k) Structural differences at the G446 loop in the BA.4 / 5 RBD are also caused by crystal contacts.(1) The 371-375 loop carrying the S371F, S373P, and S375F mutations in the BA.2.12.1 and BA.4 / 5 RBDs is stabilized by interactions with the CDR-H3 of NbC1. (m) Superposition of the BA.2.12.1 (grey), BA.2 (green, PDB 7ZF9), and BA.4 / 5 (cyan) RBDs. (n) Mutations at 452 do not introduce significant local structural changes. R452 in BA.4 / 5 has a dual conformation.

[0075] Fig.28 .Pseudovirus neutralization assay of vaccines against BA.4.6. BA.1, BA.2, BA.4.5 immune sera (ad) and monoclonal antibodies (ef). IC50 values ​​of the specified viruses obtained using sera obtained from vaccine recipients 28 days after the third dose of Pfizer BNT1 62b2 vaccine (n=22, a). IC50 values ​​of the specified viruses against 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. Analyses were performed using the Wilcoxon paired signed rank test, and two-tailed P values ​​were calculated. Neutralization curves of a panel of 28 monoclonal antibodies (e) against BA.4.6 made from samples collected from vaccine recipients infected with BA.1 were compared with Victoria, BA.1, BA.1.1, BA.2, BA.4 / 5 and BA.2.75 variants. Neutralization curves of a panel of 14 commercial monoclonal antibodies against the same variants (e). IC50 values ​​are shown in Tables 29A and 29B.

[0076] Fig.29 Pseudovirus neutralization assays. Pseudovirus neutralization assays for Omicron monoclonal antibodies associated with Table 26 where IC50 titers are shown. Neutralization curves for a panel of 27 monoclonal antibodies prepared from samples collected from vaccine recipients infected with BA.1. Titration curves for BA.2.11, BA.2.12.1 and BA.2.13 were compared to BA.2.

[0077] Fig.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) The affinity of BA.2.12.1 RBD for Omi-6 was determined using a 1:1 binding equilibrium analysis. (b), (c), (d) Sensorgrams (red: original binding curve; black: fitted curve) showing the interaction between BA.2.12.1 or BA.2 RBD and selected mAbs, with kinetic data shown.

[0078] Fig.31. Neutralization assay. Neutralization curves obtained using lentivirus pseudotyped with the S gene of the specified BA.2 sublineage (A) Omi-mAb, (B) commercial mAb. See also Table 32. The "BA.4+all" variant is a synthetic variant designed after evaluating different mutations occurring in the SARS-CoV-2 Omicron S gene. These mutations were combined and incorporated into the Omicron BA.4 S gene to produce an artificial S gene called "BA.4+all". This variant was created only as an experimental tool, does not exist in nature, and does not correspond to the S gene of any prevalent SARs-Cov-2 variant.

[0079] Fig.32 . Neutralization IC50 titers (dilution multiples) of sera pseudotyped with lentiviruses of the designated BA.2 sublineage S gene. (A) Sera obtained 28 days after the third dose of BNT162b2 vaccine or after infection with (B) BA.1, (C) BA.2 or (D) BA.4 / 5. Geometric mean titers are shown above each column. Wilcoxon paired signed rank tests (C and D) and Mann-Whitney tests (E) were used, and two-tailed P values ​​were calculated.

[0080] Fig.33 Heat map of antibody binding. The heat map shows the IC50 (μg / ml) of various antibodies against Victoria and Beta strains in vaccinated and unvaccinated samples.

[0081] Fig.34 Neutralization assay. Neutralization curves obtained using lentivirus pseudotyped with the S gene of the indicated BA.2 sublineages.

[0082] Fig.35 .Heatmap of IC50 neutralization titers for the panel of BA.1(Omi) mAbs. Pseudovirus neutralization IC50 titers of the indicated mAbs against a panel of pseudoviruses expressing variant S sequences. Live virus IC50 values ​​against variants discovered early in the pandemic are included for comparison. Live virus assay data and pseudovirus data for Victoria, BA.2, and BA.4 / 5 were previously reported in Tuekprakon et al. (2022). DETAILED DESCRIPTION

[0083] Antibodies of the present invention

[0084] The antibodies of the present invention specifically bind to the spike protein of SAR-CoV-2.

[0085] Specifically, it binds specifically to the S1 subunit of the spike protein, such as the receptor binding domain (RBD) or the N-terminal domain (NTD).

[0086] The antibody of the present invention may comprise at least three CDRs of the antibody in Table 3. The antibody may comprise at least four, five or all six CDRs of the antibody in Table 3. The antibody may comprise a heavy chain variable domain comprising 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. The antibody may comprise a light chain variable domain comprising 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. The antibody may comprise 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 the heavy chain variable domain and the light chain variable domain of the antibody in Table 3, respectively. The antibody may be any of the antibodies in Table 3.

[0087] Table 3 lists the 28 individual antibodies identified from recovered Breakthrough Omicron SARS-CoV-2 infected patients who have 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[Evidence for escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced serum]." 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 patients with recovered Beta SARS-CoV-2 infection [Liu, C et al. "The antibody response to SARS-CoV-2Beta underscores the antigenic distance to other variants". Cell hostµbe 30(1)(2021): 53-68]. The antibodies in Table 1 are also referred to herein by the prefix "COVOX", e.g., COVOX-222. The antibodies in Table 2 are also referred to herein by the prefix "β", e.g., "β50". The antibodies in Table 3 are also referred to herein by the prefix "O", e.g., "O02". Tables 1 to 3 list the nucleotide and amino acid sequences of the heavy chain variable region and light chain variable region of each antibody and the SEQ ID NO of the complementary determining region (CDR) of the variable chain.

[0088] The antibodies in Table 3 can be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38. Surprisingly, it was found that these antibodies maintained strong neutralization against live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g., IC50≤0.1 μg / ml for all live strains tested).

[0089] The antibodies in Table 3 can be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28 and Omi 08. Surprisingly, it was found that these antibodies maintained strong neutralization against live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g., IC50 ≤ 0.05 μg / ml for all live strains tested).

[0090] The antibodies in Table 3 can be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39 and Omi42. Surprisingly, it was found that these antibodies maintained strong neutralization against live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g., IC50≤0.02 μg / ml for all tested live strains).

[0091] The antibodies in Table 3 can be selected from the group consisting of: Omi03 and Omi12. Surprisingly, it was found that these antibodies maintained strong neutralization against live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g., IC50≤0.01 μg / ml for all tested live strains).

[0092] The antibodies in Table 3 can be selected from the group consisting of: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42. Surprisingly, it was found that these antibodies maintained very strong neutralization against live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron.

[0093] Thus, in one embodiment, the antibody in Table 3 may be Omi03. Omi03 was found to neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of Victoria, OmicronBA.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 invention may comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 695, 696 and 697, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 698, 699 and 700, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 692). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 694). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi03 (i.e., SEQ ID NOs: 692 and 694, respectively).

[0094] The heavy chain domain of Omi03 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi03, but not a light chain of Omi03. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 695, 696 and 697, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 692). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 692.

[0095] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi03 instead of the heavy chain of Omi03. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 698, 699 and 700, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi03 (i.e., SEQ ID NO: 694). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 694.

[0096] In one embodiment, the antibody in Table 3 can be Omi12. It was found that Omi12 can neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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, which have the amino acid sequences specified in SEQ ID NOs: 735, 736 and 737, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 738, 739 and 740, respectively. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi12 (i.e., SEQ ID NO: 732) or consisting of the amino acid sequence. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi12 (i.e., SEQ ID NO: 734) or consisting of the amino acid sequence. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi12 (i.e., SEQ ID NOs: 732 and 734, respectively).

[0097] The heavy chain domain of Omi12 is derived from the IGHV1-58 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Therefore, the antibodies of the present invention may comprise the heavy chain of Omi12, but not the light chain of Omi12. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 735, 736 and 737, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the heavy chain variable domain of antibody Omi12 (i.e., SEQ ID NO: 732). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 732.

[0098] Alternatively, in one embodiment of the present invention, the antibody may comprise a light chain of Omi12 instead of a heavy chain of Omi12. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 738, 739 and 740, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi12 (i.e., SEQ ID NO: 734). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 734.

[0099] In one embodiment, the antibody in Table 3 can be Omi02. Omi02 was found to neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 685, 686 and 687, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 688, 689 and 690, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 682). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 684). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi02 (i.e., SEQ ID NOs: 682 and 684, respectively).

[0100] The heavy chain domain of Omi02 is derived from the IGHV1-69 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise the heavy chain of Omi02, but not the light chain of Omi02. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 685, 686 and 687, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 682). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 682.

[0101] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi02 instead of the heavy chain of Omi02. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 688, 689 and 690, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi02 (i.e., SEQ ID NO: 684). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 684.

[0102] In one embodiment, the antibody in Table 3 can be Omi08. Omi08 was found to neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 715, 716 and 717, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 718, 719 and 720, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi08 (i.e., SEQ ID NO: 712). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi08 (i.e., SEQ ID NO: 714). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi08 (i.e., SEQ ID NOs: 712 and 714, respectively).

[0103] In one embodiment, the antibody in Table 3 can be Omi42. Omi42 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 955, 956 and 957, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 958, 959 and 960, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 952). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 954). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi42 (i.e., SEQ ID NOs: 952 and 954, respectively).

[0104] The heavy chain domain of Omi42 is derived from the IGHV3-9 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise the heavy chain of Omi42, but not the light chain of Omi42. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 955, 956 and 957, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 952). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 952.

[0105] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi42 instead of the heavy chain of Omi42. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 958, 959 and 960, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi42 (i.e., SEQ ID NO: 954). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 954.

[0106] In one embodiment, the antibody in Table 3 can be Omi1 6. It was found that Omi1 6 can neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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, which have the amino acid sequences specified in SEQ ID NOs: 745, 746 and 747, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 748, 749 and 750, respectively. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi16 (i.e., SEQ ID NO: 742) or consisting of the amino acid sequence. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi1 6 (i.e., SEQ ID NO: 744) or consisting of the amino acid sequence. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi16 (i.e., SEQ ID NOs: 742 and 744, respectively).

[0107] The heavy chain domain of Omi16 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Therefore, an antibody of the present invention may comprise a heavy chain of Omi16, but not a light chain of Omi16. For example, the antibody may comprise CDRH1, CDRH2, and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 745, 746, and 747, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the heavy chain variable domain of antibody Omi1 6 (i.e., SEQ ID NO: 742). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 742.

[0108] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi16 instead of a heavy chain of Omi16. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 748, 749 and 750, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi16 (i.e., SEQ ID NO: 744). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 744.

[0109] In one embodiment, the antibody in Table 3 can be Omi1 8. It was found that Omi1 8 can neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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, which have the amino acid sequences specified in SEQ ID NOs: 765, 766 and 767, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 768, 769 and 770, respectively. In one embodiment, an antibody of the present invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the heavy chain variable domain of antibody Omi18 (i.e., SEQ ID NO: 762). In one embodiment, an antibody of the present invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi18 (i.e., SEQ ID NO: 764). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi18 (i.e., SEQ ID NOs: 762 and 764, respectively).

[0110] The heavy chain domain of Omi18 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi18, but not a light chain of Omi18. For example, the antibody may comprise CDRH1, CDRH2, and CDRH3, which have the amino acid sequences set forth in SEQ ID NOs: 765, 766, and 767, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to the heavy chain variable domain of antibody Omi1 8 (i.e., SEQ ID NO: 762).

[0111] The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO:762.

[0112] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi18 instead of a heavy chain of Omi18. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 768, 769 and 770, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi18 (i.e., SEQ ID NO: 764). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 764.

[0113] In one embodiment, the antibody in Table 3 can be Omi20. Omi20 was found to neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 775, 776 and 777, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 778, 779 and 780, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 772). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 774). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi20 (i.e., SEQ ID NOs: 772 and 774, respectively).

[0114] The heavy chain domain of Omi20 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi20, but not a light chain of Omi20. For example, the antibody may comprise CDRH1, CDRH2, and CDRH3, which have the amino acid sequences set forth in SEQ ID NOs: 775, 776, and 777, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity to the heavy chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 772). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 772.

[0115] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi20 instead of a heavy chain of Omi20. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 778, 779 and 780, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi20 (i.e., SEQ ID NO: 774). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 774.

[0116] In one embodiment, the antibody in Table 3 can be Omi23. Omi23 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 785, 786 and 787, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 788, 789 and 790, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 782). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 784). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi23 (i.e., SEQ ID NOs: 782 and 784, respectively).

[0117] The heavy chain domain of Omi23 is derived from the IGHV4-31 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi23, but not a light chain of Omi23. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 785, 786 and 787, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 782). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 782.

[0118] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi23 instead of the heavy chain of Omi23. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 788, 789 and 790, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi23 (i.e., SEQ ID NO: 784). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 784.

[0119] In one embodiment, the antibody in Table 3 can be Omi28. Omi28 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 835, 836 and 837, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 838, 839 and 840, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 832). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 834). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to 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 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the invention may comprise the heavy chain of Omi28, but not the light chain of Omi28. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences set forth in SEQ ID NOs: 835, 836 and 837, respectively.The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 832). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 832.

[0120] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi28 instead of a heavy chain of Omi28. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 838, 839 and 840, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi28 (i.e., SEQ ID NO: 834). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 834.

[0121] In one embodiment, the antibody in Table 3 can be Omi39. Omi39 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 935, 936 and 937, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 938, 939 and 940, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi39 (i.e., SEQ ID NO: 932). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi39 (i.e., SEQ ID NO: 934). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi39 (i.e., SEQ ID NOs: 932 and 934, respectively).

[0122] In one embodiment, the antibody in Table 3 can be Omi17. It was found that Omi17 can neutralize live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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, which have the amino acid sequences specified in SEQ ID NOs: 755, 756 and 757, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 758, 759 and 760, respectively. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi17 (i.e., SEQ ID NO: 752) or consisting of the amino acid sequence. In one embodiment, the antibody of the present invention may comprise 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 antibody Omi1 7 (i.e., SE0 ID NO: 754) or consisting of the amino acid sequence. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi17 (i.e., SEQ ID NOs: 752 and 754, respectively).

[0123] The heavy chain domain of Omi17 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for the present invention (further explained below). Therefore, the antibodies of the present invention may comprise the heavy chain of Omi17, but not the light chain of Omi17. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 755, 756 and 757, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the heavy chain variable domain of antibody Omi1 7 (i.e., SEQ ID NO: 752). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 752.

[0124] Alternatively, in one embodiment of the present invention, the antibody may comprise a light chain of Omi17 instead of a heavy chain of Omi17. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 758, 759 and 760, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi17 (i.e., SEQ ID NO: 754). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 754.

[0125] In one embodiment, the antibody in Table 3 can be Omi29. Omi29 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 845, 846 and 847, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 848, 849 and 850, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 842). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 844). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi29 (i.e., SEQ ID NOs: 842 and 844, respectively).

[0126] The heavy chain domain of Omi29 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi29, but not a light chain of Omi29. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences set forth in SEQ ID NOs: 845, 846 and 847, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 842). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 842.

[0127] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi29 instead of the heavy chain of Omi29. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 848, 849 and 850, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi29 (i.e., SEQ ID NO: 844). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 844.

[0128] In one embodiment, the antibody in Table 3 can be Omi36. Omi36 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 915, 916 and 917, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 918, 919 and 920, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 912). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 914).

[0129] In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to 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 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the invention may comprise the heavy chain of Omi36, but not the light chain of Omi36. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences set forth in SEQ ID NOs: 915, 916 and 917, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 912). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 912.

[0130] Alternatively, in one embodiment of the invention, the antibody may comprise the light chain of Omi36 instead of the heavy chain of Omi36. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 918, 919 and 920, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Omi36 (i.e., SEQ ID NO: 914). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 914.

[0131] In one embodiment, the antibody in Table 3 can be Omi38. Omi38 was found to neutralize live SARS-CoV-2 variants Victoria, Alpha, Beta, Gamma, Delta and Omicron. and pseudovirus constructs of 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 comprise: CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 925, 926 and 927, respectively; and CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 928, 929 and 930, respectively. In one embodiment, an antibody of the invention may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi38 (i.e., SEQ ID NO: 922). In one embodiment, an antibody of the invention may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of antibody Omi38 (i.e., SEQ ID NO: 924). In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain and light chain variable domain of antibody Omi38 (i.e., SEQ ID NOs: 922 and 924, respectively).

[0132] The heavy chain domain of Omi38 is derived from the IGHV1-69 v-region, and the inventors have previously demonstrated that switching heavy and light chains between antibodies derived from the same v-region can produce antibodies that are particularly suitable for use in the present invention (further explained below). Thus, an antibody of the present invention may comprise a heavy chain of Omi38, but not a light chain of Omi38. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3, which have the amino acid sequences specified in SEQ ID NOs: 925, 926 and 927, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi38 (i.e., SEQ ID NO: 922). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 922.

[0133] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi38 instead of a heavy chain of Omi38. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3, which have the amino acid sequences specified in SEQ ID NOs: 928, 929 and 930, respectively. The antibody may comprise a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the light chain variable domain of antibody Om38 (i.e., SEQ ID NO: 924). The antibody may comprise a light chain variable domain comprising or consisting of SEQ ID NO: 924.

[0134] Mixed chain antibodies of the present invention

[0135] The antibodies of the present invention may comprise a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 of a first antibody from Table 1, 2 or 3, and a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 of a second antibody from Table 1, 2 or 3, provided that the first antibody and the second antibody are different. Such antibodies are referred to herein as mixed chain antibodies.

[0136] Examples of mixed chain antibodies that can be used in the present invention are provided in Tables 4 to 12. Table 4 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV 3-53 produced by the antibodies in Tables 1 to 3. Table 5 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV 3-53 and IGHV3-66 produced by the antibodies in Tables 1 to 3. Table 6 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV1-58 produced by the antibodies in Tables 1 to 3. Table 7 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV1-69 produced by the antibodies in Tables 2 and 3. Table 8 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV3-30 produced by the antibodies in Tables 1 to 3. Table 9 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV3-33 produced by the antibodies in Tables 2 and 3. Table 10 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV1-18 produced by the antibodies in Tables 1 to 3. Table 11 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV3-9 produced by the antibodies in Tables 1 and 3. Table 12 shows examples of mixed chain antibodies derived from the same germline heavy chain IGHV4-31 produced by the antibodies in Tables 2 and 3. Examples of mixed chain antibodies derived from the same germline heavy chain IGHV1-69 are Omi02H / Beta-49L and Omi38H / Omi24L.

[0137] Thus, in one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 of a first antibody from Table 1, 2 or 3, and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 of a second antibody from Table 1, 2 or 3, with the proviso that the first antibody and the second antibody are different. The antibody may comprise a heavy chain variable domain amino acid sequence having at least 80% sequence identity with the heavy chain variable domain of the first antibody from 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 of the second antibody from Table 1, 2 or 3, with the proviso that the first antibody and the second antibody are different. For example, the antibody may comprise a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of an antibody in Table 1, 2 or 3, and the light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of an antibody in Table 1, 2 or 3, with the proviso that the first antibody and the second antibody are different.

[0138] The first antibody may be in Table 3, and the second antibody may be in Table 3.

[0139] The first antibody may be in Table 3, and the second antibody may be in Table 1. The first antibody may be in Table 3, and the second antibody may be in Table 2. The first antibody may be in Table 1, and the second antibody may be in Table 3. The first antibody may be in Table 2, and the second antibody may be in Table 3. The first antibody may be in Table 1, and the second antibody may be in Table 2. The first antibody may be in Table 2, and the second antibody may be in Table 1. The first antibody may be in Table 2, and the second antibody may be in Table 2. The first antibody may be in Table 1, and the second antibody may be in Table 1.

[0140] In one embodiment, at least one of the first antibody and the second antibody is an antibody from Table 3.

[0141] In one embodiment, the first antibody and the second antibody are not both in Table 1. In one embodiment, the first antibody and the second antibody are not both in Table 2. In one embodiment, the first antibody and the second antibody are not both selected from antibodies in Table 1 or 2.

[0142] In one embodiment, at least one of the heavy chain variable domain and the light chain variable domain is from Table 3.

[0143] The antibodies in Table 3 may be selected from the group consisting of: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42. The antibodies in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, 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 in Table 3 may be selected from the group consisting of: Omi03 and Omi12.

[0144] In one embodiment, the first antibody and the second antibody are both 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. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% sequence identity with the corresponding variable domain of any mixed chain antibody as listed in Table 4.

[0145] Antibodies derived from IGHV3-53 can be used to generate mixed-chain antibodies with antibodies from IGHV3-66 (e.g., antibodies 40 and 398 in Table 1) (see, 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 [Evidence for escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced serum]." 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, the first antibody and the second antibody are both 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 IGVH3-66.The resulting mixed chain antibodies are listed in Table 5. Thus, the antibodies of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or a heavy chain variable domain and a light chain variable domain, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domain of any one of the mixed chain antibodies listed in Table 5.

[0146] In one embodiment, the first antibody and the second antibody are both 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 IGHV1-58. The resulting mixed chain antibodies are listed in Table 6. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domain of any mixed chain antibody listed in Table 6 or consisting of the amino acid sequence.

[0147] In one embodiment, the first antibody and the second antibody are both 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. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domains of any mixed chain antibody as listed in Table 7.

[0148] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of Beta-22, Beta-29, Antibody 159 and Omi09. The heavy chain variable domains of these antibodies are derived from IGHV 3-30. The resulting mixed chain antibodies are listed in Table 8. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domains of any mixed chain antibody as listed in Table 8.

[0149] In one embodiment, the first antibody and the second antibody are both 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. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domains of any mixed chain antibody as listed in Table 9. The CDRL1-3 of Omi32 and Omi33 are identical, which means that they are actually exemplary mixed chain antibodies of the present invention.

[0150] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of antibody 278, Beta-44, Omi26 and Omi41. The heavy chain variable domains of these antibodies are derived from IGHV 1-18. The resulting mixed chain antibodies are listed in Table 10. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domains of any mixed chain antibody as listed in Table 10.

[0151] In one embodiment, the first antibody and the second antibody are both 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 listed in Table 11. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domain of any mixed chain antibody as listed in Table 11.

[0152] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of Beta-56 and Omi23. The heavy chain variable domains of these antibodies are derived from IGHV 4-31. The resulting mixed chain antibodies are listed in Table 12. Therefore, the antibodies of the present invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and / or heavy chain variable domains and light chain variable domains, each of which comprises or consists of an amino acid sequence having at least 80% (e.g., ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity with the corresponding variable domains of any mixed chain antibody listed in Table 12.

[0153] The constant region domains (if present) of the antibody molecules of the present invention can be selected taking into account the intended function of the antibody molecule, in particular the effector functions that may be required. For example, the constant region domains can be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant region is of human origin. Specifically, human IgG (i.e., IgG1, IgG2, IgG3 or IgG4) constant region domains can be used. Typically, the constant region is a human IgG1 constant region.

[0154] Certain Antibodies of the Invention

[0155] The present invention also provides an antibody which is a full-length antibody of any one of the antibodies in Tables 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In other words, the antibody of the present invention comprises a heavy chain variable domain and a light chain variable domain, which are respectively composed of the heavy chain variable domain and the 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.

[0156] For example, the antibodies of the present invention may be full-length Omi02, Omi03, Omi12, Omi1 8, Omi28, Omi39 or Omi42 antibodies. The antibodies of the present invention may be full-length Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 or Omi38 antibodies. These antibodies are highly effective neutralizing mAbs that have been shown to neutralize the Omicron variant of SARS-CoV-2 with an IC50 ≥ 0.1 μg / ml. These antibodies also specifically neutralize at least Victoria, Alpha, Beta, Gamma and Delta strains of SARS-CoV-2 with an IC50 ≤ 0.1 μg / ml.

[0157] The antibody may be derived from the germline heavy chain IGHV1-58 and comprise a proline at position 53 in the heavy chain variable region (according to absolute numbering). For example, the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to 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 in the heavy chain variable region is a proline (according to absolute numbering). For example, the antibody may comprise the heavy 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 for the V53P mutation in the heavy chain variable region. The inventors have found that such antibodies are particularly effective against the Omicron strain (see, e.g., Example 5).

[0158] Position 53 of the heavy chain variable regions of IGHV1-58-derived antibodies Omi-12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253, and Antibody 318 corresponds to position 58 according to IMGT numbering.

[0159] Therefore, the present invention also provides an antibody derived from the germline heavy chain IGHV1-58, which is capable of binding to the spike protein of the coronavirus SARS-CoV-2, wherein the amino acid at position 58 in the heavy chain variable region according to IMGT numbering is proline or is substituted with proline.

[0160] The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of an antibody derived from germline heavy chain IGHV1-58, with the proviso that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline.

[0161] The antibody 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 (e.g., see Tables 1 to 3). The amino acid sequence of the heavy chain variable domain of antibody AZD8895 is provided in SEQ ID NO: 963.

[0162] The IGHV1-58 germline V gene sequence encodes the following amino acid sequence: MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRL EWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSED TAVYYCAA (SEQID NO: 961). Therefore, the present invention also provides an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, the antibody comprising a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence having ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO: 961, provided that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline.

[0163] The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963, with the proviso that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline. The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 591, 461, 62, 182, 262 or 332, wherein the valine at position 58 according to IMGT numbering is substituted with proline.

[0164] The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 963, wherein the isoleucine at position 58 according to IMGT numbering is substituted with proline.

[0165] In some embodiments, an antibody derived from germline heavy chain IGHV1-58 comprises a light chain variable domain derived from IGLV Kappa 3-20. The antibody may comprise a light chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to a light chain variable domain of an antibody derived from germline IGLV Kappa 3-20. The germline IGLV Kappa 3-20 V sequence may encode the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSV SSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO: 967). Thus, an antibody derived from germline heavy chain IGHV1-58 may comprise a light chain variable domain comprising an amino acid sequence having ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO:967.

[0166] The present invention also provides an antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, the antibody comprising a heavy chain variable domain, the heavy chain variable domain comprising 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 is substituted with proline. The modified version of SEQ ID NO: 961 may comprise modifications as described herein, such as substitutions, deletions and / or additions. For example, the modification may include ≤50, ≤45, ≤40, ≤35, ≤30, ≤25, ≤20, ≤15, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2 or 1 amino acid substitution and / or deletion of SEQ ID NO: 961. The modification may include ≤4, ≤3, ≤2 or 1 amino acid substitution and / or deletion of SEQ ID NO: 961.

[0167] The antibody may comprise a heavy chain variable domain comprising an amino acid sequence that is a modified version of SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963, comprising ≤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 is substituted with proline. The modified version of SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963 may comprise modifications as described herein, e.g., substitutions, deletions and / or additions.

[0168] The antibody can comprise an IgG (eg, IgG1) constant region.

[0169] The present invention also provides a method for preparing such antibodies. For example, the method may include modifying an antibody derived from germline heavy chain IGHV1-58 by replacing the amino acid at position 58 (according to IMGT numbering) in the heavy chain variable region with proline, which antibody is capable of engaging with the spike protein of coronavirus SARS-CoV-2. The antibody derived from germline heavy chain IGHV1-58 can be AZD8895, Omi-12, Beta-47, Beta-25, antibody 55, antibody 165, antibody 253 or antibody 318. The amino acid sequence of the heavy chain variable domain of each of these antibodies is described herein (e.g., see Tables 1 to 3 and SEQ ID NO: 963). The present invention also provides an antibody obtainable or obtained by this method.

[0170] Characteristics of the Antibodies of the Invention

[0171] The antibodies of the present invention may be or may comprise modifications of the amino acid sequences of the antibodies from Tables 1 to 12 while maintaining the activity and / or function of the antibodies. The modifications may be substitutions, deletions and / or additions. For example, the modifications may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions of the amino acid sequences of the antibodies from Tables 1 to 12. For example, the modifications may comprise amino acids substituted with alternative amino acids having similar properties. Some properties of the 20 major amino acids that can be used to select suitable substitutes are as follows:

[0172] Ala Aliphatic, hydrophobic, neutral Met Hydrophobic, neutral Cys Polar, hydrophobic, neutral Asn Polar, hydrophilic, neutral Asp Polar, hydrophilic, charged (-) Pro Hydrophobic, neutral Glu Polar, hydrophilic, charged (-) Gln Polar, hydrophilic, neutral Phe Aromatic, hydrophobic, neutral Arg Polar, hydrophilic, charged (+) Gly Aliphatic, neutral Ser Polar, hydrophilic, neutral His Aromatic, polar, hydrophilic, charged (+) Thr Polar, hydrophilic, neutral Ile Aliphatic, hydrophobic, neutral Val Aliphatic, hydrophobic, neutral Lys Polar, hydrophilic, charged (+) Trp Aromatic, hydrophobic, neutral Leu Aliphatic, hydrophobic, neutral Tyr Aromatic, polar, hydrophobic

[0173] Such modifications may include derivatized amino acids, such as labeled or non-natural amino acids, provided that the function of the antibody is not significantly adversely affected.

[0174] Modifications of the antibodies of the invention as described above can be made during the synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form, using known site-directed mutagenesis, random mutagenesis or nucleic acid enzymatic cleavage and / or ligation techniques.

[0175] The antibodies of the present invention may be modified (e.g., as described above) to improve the efficacy of the antibodies or to adapt the antibodies to new SARS-CoV-2 variants. These modifications may be amino acid substitutions to adapt the antibodies to substitutions in viral variants. For example, the known binding mode of the antibody to the spike protein (e.g., determined by crystal structure or modeling) can be used to identify amino acids of the antibody that interact with substitutions in viral variants. This information can then be used to identify possible substitutions of the antibody that will compensate for changes in epitope characteristics. For example, substitutions of hydrophobic amino acids in the spike protein with negatively charged amino acids can be compensated by substitutions of amino acids from the antibody that interact with the amino acids in the spike protein with positively charged amino acids. The present disclosure includes methods for identifying residues of an antibody that can be substituted, for example, by determining the structure of an antibody-antigen complex as described herein.

[0176] The antibodies of the present invention may contain one or more modifications to increase their cross-lineage neutralization properties. For example, E484 of the spike protein (a key residue that mediates interaction with ACE2) mutates in some SARS-CoV-2 strains (e.g., the Victoria strain contains E484, but the P.1 and B.1.351 strains contain E484K), resulting in different neutralizing effects of the antibodies. Therefore, antibodies that bind to E484 can be modified to compensate for changes in E484 of the spike protein. For example, when compared to the original strain, E484 mutates from a positively charged amino acid to a negatively charged amino acid in SAR-CoV-2 strains of the B.1.351 or P.1 lineages. The amino acid residues that bind to or are close to E484 of the antibody may mutate 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.

[0177] The antibodies of the present invention may be isolated antibodies. An isolated antibody is an antibody that is substantially free of other antibodies having different antigenic specificities.

[0178] The term "antibody" as used herein may relate to a complete antibody (i.e., an element comprising two heavy chains and two light chains interconnected by disulfide bonds) as well as an antigen-binding fragment thereof. Antibodies generally comprise an immunologically active portion of an immunoglobulin (Ig) molecule (i.e., a molecule containing an antigen binding site that specifically binds to (immunoreacts with) an antigen). By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and at least one heavy chain constant region. Each light chain is composed 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 hypervariable regions known as complementarity determining regions (CDRs), with more conservative regions known as framework regions (FRs) inserted therebetween.

[0179] 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.

[0180] The antibodies of the present invention may be monoclonal antibodies. The monoclonal antibodies (mAbs) of the present invention may be produced by various techniques, including conventional monoclonal antibody methods, such as those disclosed in "Monoclonal Antibodies: a manual of techniques" (Zola H, 1987, CRC Press [Press]) and "Monoclonal Hybridoma Antibodies: techniques and applications" (Hurrell JGR, 1982 CRC Press [Press]). The antibodies of the present invention may be multispecific, such as bispecific. The bispecific antibodies of the present invention bind to two different epitopes. These epitopes may be in the same protein (e.g., two epitopes in the spike protein of SARS-CoV-2) or in different proteins (e.g., one epitope in the spike protein of SARS-CoV-2 and one epitope in another protein (such as the coat protein)).

[0181] In one embodiment, the bispecific antibodies of the invention can bind to two separate epitopes on the spike protein of SARS-CoV-2. The bispecific antibodies can bind to the NTD of the spike protein and the RBD of the spike protein. The bispecific antibodies can bind to two different epitopes in the RBD of the spike protein.

[0182] One or more (eg, two) antibodies of the invention can be coupled to form a multispecific (eg, bispecific) antibody. Methods for preparing multispecific (eg, bispecific) antibodies are well known in the art.

[0183] The antibody can 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 comprising the antigen-binding domain of natural antibodies or aptamers, single domain antibodies (sdAb) (also known as VHH antibodies), nanobodies (single domain antibodies derived from Camelid), single domain antibody fragments derived from shark IgNAR (called VNARs), diabodies, triabodies, Anticalins, aptamers (DNA or RNA), and active components or fragments thereof.

[0184] The constant region domains (if present) of the antibody molecule of the present invention can be selected taking into account the intended function of the antibody molecule, in particular the effector functions that may be required. For example, the constant region domains can be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant region is of human origin. Specifically, human IgG (i.e., IgG1, IgG2, IgG3 or IgG4) constant region domains can be used. Typically, the constant region is a human IgG1 constant region.

[0185] The light chain constant region can be lambda or kappa.

[0186] The antibodies of the invention may be monospecific or multispecific (eg, bispecific).Multispecific antibodies contain at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or a different epitope on the same antigen.

[0187] The antibody of the present invention can be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human antibody or a humanized antibody. Typically, the antibody is a human antibody. A fully human antibody is one in which the variable regions and constant regions (where present) of the heavy and light chains are of human origin or are substantially identical to sequences of human origin but not necessarily from the same antibody.

[0188] The antibodies of the present invention may be full-length antibodies.

[0189] The antibodies of the present invention can be antigen binding fragments. The antigen binding fragments of the present invention bind to the same epitope of the parent antibody (i.e., the antigen binding fragments are derived from and the antibodies therein). The antigen binding fragments of the present invention generally retain the part of the parent antibody that interacts with the epitope. The antigen binding fragments generally comprise a complementary determining region (CDR) that interacts with the antigen, such as one, two, three, four, five or six CDRs. In certain embodiments, the antigen binding fragments further comprise a structural scaffold surrounding the CDRs of the parent antibody, such as the variable region domains of the heavy chain and / or light chain. Typically, the antigen binding fragment retains the same or similar binding affinity to the antigen as the parent antibody.

[0190] The antigen binding fragment does 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%, 100% sequence identity with the corresponding CDR of the parent antibody. In one embodiment, the antigen binding fragment may have ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity with the corresponding variable region domain of the parent antibody. Typically, the non-identical amino acids in the variable region are not in the CDR.

[0191] The antigen-binding fragment of the antibody of the present invention retains the ability to selectively bind to the antigen. Antigen-binding fragments of antibodies include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv.

[0192] The antigen binding function of an antibody can be achieved by a fragment of a full-length antibody. Methods for generating and manufacturing these antibody fragments are well known in the art (see, e.g., Verma R et al., 1998, J. Immunol. Methods, 216, 165-181).

[0193] Methods for screening antibodies of the invention that do not have 100% amino acid sequence identity to one of the antibodies disclosed herein, that have the desired specificity, affinity, and functional activity include methods described herein, such as enzyme-linked immunosorbent assay, biacore, focus reduction neutralization assay (FRNT), and other techniques known in the art.

[0194] Regarding function, the antibodies of the invention may be able to neutralize at least one biological activity of SAR-CoV-2 (neutralizing antibodies), in particular neutralizing viral infectivity.

[0195] Neutralization can also be determined using IC50 or IC90 values. For example, the antibody may have an IC50 value 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 invention may have an IC50 value between 0.0001 μg / ml and 0.1 μg / ml, sometimes between 0.0001 μg / ml and 0.05 μg / ml, or even between 0.0001 μg / ml and 0.001 μg / ml.

[0196] For example, IC50 values ​​for some of the antibodies in Tables 1 to 12 are provided in Tables 13 to 16.

[0197] The ability of an antibody to neutralize viral infectivity can be measured using an appropriate assay, in particular using a cell-based neutralization assay, as shown in the Examples. For example, the neutralization capacity can be measured in a focus reduction neutralization assay (FRNT), where the reduction in the number of cells (e.g., human cells) infected with the virus (e.g., infected for 2 hours at 37° C.) in the presence of an antibody is compared to a negative control to which no antibody is added.

[0198] The antibodies of the present invention can block the interaction between the spike protein of SAR-CoV-2 and the cell surface receptor angiotensin-converting enzyme 2 (ACE2) of the target cell, for example by directly blocking or by disrupting the pre-fusion conformation of the spike protein.

[0199] The blocking of the interaction between the spike protein and ACE2 can be complete or partial. For example, the antibodies of the present invention can reduce the formation of spike protein-ACE2 by ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100%. The blocking of the formation of spike protein-ACE2 can be measured by any suitable means known in the art, for example, by EIISA.

[0200] Most of the antibodies that showed neutralization were also shown to block the interaction between the spike protein and ACE2. In addition, many non-neutralizing antibodies were good ACE2 blockers.

[0201] In terms of binding kinetics, the antibodies of the invention may have an affinity constant (KD) value for the spike protein of SARS-CoV-2 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.

[0202] KD values ​​can be measured by any suitable means known in the art, for example, by ELISA or surface plasmon resonance (Biacore) at 25°C.

[0203] Binding affinity (KD) can be quantified by determining the dissociation constant (Kd) and association constant (Ka) of an antibody and its target. For example, the antibody may have a KD of ≥10,000 M. -1 s -1 ,≥50000M -1 s -1 ,≥100000M -1 s -1 ,≥200000M -1 s -1 or ≥500000M -1 s -1 Association constant (Ka) and / or ≤0.001s -1 ,≤0.0005s -1 ,≤0.004s -1 ,≤0.003s -1 ,≤0.002s -1 or ≤0.0001s -1 The dissociation constant (Kd) of

[0204] The antibodies of the present invention are preferably able to provide in vivo protection in animals infected with coronavirus (e.g., SARS-CoV-2). For example, administration of the antibodies of the present invention to animals infected with coronavirus (e.g., SARS-CoV-2) can result in a survival rate of ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or 100%. Survival rates can be determined using conventional methods.

[0205] The antibodies of the present invention may have any combination of one or more of the above-mentioned properties.

[0206] The antibodies of the invention may bind to the same epitope as any of the antibodies described herein (i.e., particularly antibodies having the above-described heavy and light chain variable regions), or compete with any of the antibodies described herein for binding to the SARS-CoV-2 spike protein. Methods for identifying antibodies that bind to the same epitope or cross-compete with each other are used in the Examples and are discussed further below.

[0207] Fc region

[0208] The antibodies of the invention may or may not comprise an Fc domain.

[0209] The antibodies of the present invention may be modified in the Fc region to improve their stability. Such modifications are known in the art. Modifications may improve the stability of the antibody during storage of the antibody. The in vivo half-life of the antibody may be improved by modifying the Fc region. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in the region. The homodimeric antibodies thus produced may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med. [Journal of Experimental Medicine], 176: 1191-1195 (1992) and Shopes, J. Immunol. [Journal of Immunology], 148: 2918-2922 (1992)).

[0210] Alternatively, antibodies with dual Fc regions can be engineered, thereby enhancing complement lysis and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0211] For example, the antibodies of the present invention can be modified to promote the interaction of the Fc domain with FcRn. The Fc domain can be modified to improve the stability of the antibody by affecting the interaction of Fc and FcRn at low pH (such as in endosomes). The M252Y / S254T / T256E (YTE) mutation can be used to improve the half-life of IgG1 antibodies.

[0212] The antibodies may be modified to affect the interaction of the antibody with other receptors such as FcγRI, FcγRIIA, FcγRIIB, FcγRIII and FcαR. Such modifications may be used to affect the effector functions of the antibodies.

[0213] In one embodiment, the antibody of the invention comprises an altered Fc domain as described below. In another preferred embodiment, the antibody of the invention comprises an Fc domain, but the sequence of the Fc domain has been altered to modify one or more Fc effector functions.

[0214] In one embodiment, the antibodies of the invention comprise a "silent" Fc region. For example, in one embodiment, the antibodies of the invention do not exhibit effector functions or functions associated with a normal Fc region. The Fc region of the antibodies of the invention does not bind to one or more Fc receptors.

[0215] In one embodiment, the antibodies of the invention do not comprise a CH2 domain. In one embodiment, the antibodies of the invention do not comprise a CH3 domain. In one embodiment, the antibodies of the invention comprise additional CH2 and / or CH3 domains.

[0216] In one embodiment, the antibodies of the invention do not bind Fc receptors. In one embodiment, the antibodies of the invention do not bind complement. In an alternative embodiment, the antibodies of the invention do not bind FcγRs, but do bind complement.

[0217] In one embodiment, antibodies of the present invention generally may comprise modifications that change the serum half-life of the antibody. Thus, in another embodiment, antibodies of the present invention have Fc region modifications that change the half-life of the antibody. Such modifications may exist as those that change Fc function. In a preferred embodiment, antibodies of the present invention have modifications that change the serum half-life of the antibody.

[0218] In one embodiment, the antibodies of the present invention may comprise human constant regions, such as IgA, IgD, IgE, IgG or IgM domains. Specifically, when the antibody molecule is intended for therapeutic use requiring antibody effector functions, human IgG constant region domains, especially IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is used for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes may be used.

[0219] In one embodiment, the antibody heavy chain comprises a CH1 domain, and the antibody light chain comprises a CL domain (κ or λ). In one embodiment, the antibody heavy chain comprises a CH1 domain, a CH2 domain, and a CH3 domain, and the antibody light chain comprises a CL domain (κ or λ).

[0220] The four human IgG isotypes bind with different affinities to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa), inhibitory FcγRIIb receptors and the first component of complement (C1 q), 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 antibodies of the invention do not bind to Fc receptors. In another embodiment of the invention, the antibodies do bind to one or more types of Fc receptors.

[0221] In one embodiment, the Fc region employed is a mutation, in particular a mutation as described herein. In one embodiment, the Fc mutation is selected from the group comprising: a mutation that removes or enhances binding of the Fc region to an Fc receptor, a mutation that adds or removes effector function, a mutation that increases or decreases the half-life of the antibody, and combinations thereof. In one embodiment, when the effect of a modification is mentioned, it can be demonstrated by comparison with an equivalent antibody lacking the modification.

[0222] Some antibodies that selectively bind FcRn at pH 6.0 but not 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 inprimates. 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 the binding affinity to FcRn and the half-life of IgG1 in vivo. Thus, modifications that alter serum half-life may be present at M252 / S254 / T256+H44 / N434, in particular M252Y / S254T / T256E+H433K / N434F may be present. In one embodiment, it is desirable to increase half-life. In another embodiment, it may actually be desirable to reduce the serum half-life of the antibody, and therefore modifications that reduce serum half-life may be present.

[0223] A large number of mutations have been made in the CH2 domain of human IgG1 and their effects on ADCC and CDC have been tested in vitro (Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4): 2571-5). Of note, it has been reported that alanine substitution at position 333 can increase ADCC and CDC. Thus, in one embodiment, a modification may be present at position 333, particularly one that alters the ability to recruit complement. Lazar et al. described a triple mutant (S239D / I332E / A330L) that has a higher affinity for FcγRIIIa and a lower affinity for FcγRIIb, thereby enhancing ADCC (Lazar GA. et al., 2006). Thus, modifications may be present at S239 / I332 / A330, particularly those that alter affinity for Fc receptors, particularly S239D / I332E / A330L. Engineered antibody Fc variants with enhanced effector function. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 103(11): 4005-4010). The same mutations were used to generate antibodies with increased ADCC (Ryan MC. et al., 2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer Ther [Molecular Cancer Therapy], 6: 3009-3018). Richards et al. studied a slightly different triple mutant (S239D / I332E / G236A) with increased FcγRIIIa affinity and FcγRIIa / FcγRIIb ratio, which mediates enhanced macrophage phagocytosis of target cells (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). In one embodiment, the S239D / I332E / G236A modification may therefore be present.

[0224] In another embodiment, the antibodies of the invention may have a modified hinge region and / or CH1 region. Alternatively, the isotype employed may be selected because it has a particular hinge region.

[0225] Main public area

[0226] Common V regions (also described herein as common V genes) are the V regions of the germline heavy and light chain regions found in the majority of antibody responses to SARS-CoV-2 in a population. In this application, the V regions are specifically reactive to the Beta SARS-CoV-2 variant. That is, many individuals will utilize the same V regions from their germline V region repertoire when generating an immune response to a SARS-CoV-2 variant.

[0227] As used herein, an antibody "derived from" a specific v region refers to an antibody produced by V(D)J recombination using the germline v region sequence. For example, the germline IGHV3-53 v region sequence can undergo somatic recombination and somatic mutation to produce an antibody that specifically binds to the spike protein of SARS-CoV-2. The nucleotide sequence encoding the antibody is unlikely to contain a sequence identical to the IGHV3-53 germline sequence, but the antibody is still derived from the v region. When compared to the germline sequence, the antibodies of the present invention typically contain only non-silent mutations in the v region, such as no more than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 non-silent mutations. When compared to the germline sequence, the antibodies of the present invention typically do not contain 2-20 non-silent mutations in the v region, such as 5-15, 6-13 and 7-12 non-silent mutations. Germline v region sequences are well known in the art, and methods for identifying whether a certain region of an antibody is derived from a specific germline v region sequence are also well known in the art.

[0228] In one embodiment, the antibodies of the invention are 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 found that the potent neutralizing antibodies identified herein contain relatively few mutations in the CDRs of these v regions. Thus, in one embodiment, the antibodies of the invention are encoded by the following v regions: selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV1-69, IGHV3-30, IGHV3-33, IGHV1-18, IGHV13-9 or IGHV4-31, and have 2-20 non-silent nucleotide mutations or 5-15 non-silent mutations when compared to the naturally occurring germline sequence, such as 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 non-silent mutations. Silent mutations as defined herein are changes in the nucleotide sequence, while the amino acid sequence encoded by the nucleotide sequence does not change. Therefore, non-silent mutations are mutations that result in changes in the amino acid sequence encoded by the nucleotide sequence.

[0229] The inventors were surprised to find that the light chain variable regions of two antibodies with the same heavy chain v region can be exchanged to produce a mixed chain antibody comprising the heavy chain variable region of the first antibody and the light chain variable region of the second antibody. For example, both antibodies may comprise a heavy chain variable region derived from IGHV3-53. Preferably, the two antibodies also comprise a light chain variable region derived from the same light chain v region, although this is not necessary, because, for example, the light chain of antibody 222 can match any heavy chain variable region derived from IGHV3-53 and produce potent neutralizing antibodies. As described above, the two antibodies may comprise a heavy chain variable region derived from IGHV3-53 and / or IGHV3-66.

[0230] In one embodiment, an antibody of the invention comprises a CDR of a heavy chain variable domain of an antibody selected from the major common v-regions of IGHV3-53, IGHV1-58, IGHV3-66, IGHV4-39, IGHV3-30, IGHV5-51, IGHV1-02 or IGHV3-33, such as, for IGHV3-53, antibodies Omi03, Omi1 8, 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 55, antibody 165, and antibody 253 for IGHV1-69, antibodies Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34, and Omi38 for IGHV3-30, antibodies Beta-22, Beta-29, antibody 159, and Omi09 for IGHV3-33, antibodies Beta-20, Beta-43, Omi32, and Omi 33, for IGHV1-18 it is antibody 278, Beta-44, Omi26 and Omi41, for IGHV3-9 it is antibody 58, Omi25, Omi35 and Omi42, or for IGHV4-31 it is antibody Beta-56 and Omi23. The SEQ ID NO corresponding to the CDR of each of these antibodies is shown in Tables 1, 2 and 3.

[0231] In one embodiment, an antibody of the invention comprises a heavy chain variable domain derived from an antibody selected from the major common v region of IGHV3-53, IGHV1-58, IGHV3-66, IGHV4-39, IGHV3-30, IGHV5-51, IGHV1-02 or IGHV3-33, such as, for IGHV3-53, antibodies Omi03, Omi1 8, 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 55, antibody 165, antibody 253 for IGHV1-69, antibodies Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34 and Omi38 for IGHV3-30, antibodies Beta-22, Beta-29, antibody 159 and Omi09 for IGHV3-33, antibodies Beta-20, Beta-43, Omi32 and Omi 33, for IGHV1-18 it is antibody 278, Beta-44, Omi26 and Omi41, for IGHV3-9 it is antibody 58, Omi25, Omi35 and Omi42, or for IGHV4-31 it is antibody Beta-56 and Omi23. The SEQ ID NO corresponding to the CDR of each of these antibodies is shown in Tables 1, 2 and 3.

[0232] In one embodiment, the present invention provides a method for producing an antibody that specifically binds to a spike protein of SARS-CoV-2 (e.g., a SARS-CoV-2 strain of the Alpha, Beta, Gamma, Delta, and / or Omicron lineage), the method comprising identifying two or more antibodies derived from the same light chain and / or heavy chain v region, replacing the light chain of the first antibody with the light chain of the second antibody, thereby producing a mixed chain antibody comprising the heavy chain of the first antibody and the light chain of the second antibody. In one embodiment, the method further comprises determining the affinity and / or neutralization of the mixed chain antibody to SARS-CoV-2. The method may also comprise comparing the affinity of the mixed chain antibody to the affinity of the first and / or second antibody. The method may also comprise selecting a mixed chain antibody having the same affinity as the first and / or second antibody or having a greater affinity than them. In some embodiments, the heavy chain v region is IGHV 1-58, and / or the light chain v region is IGLV Kappa 3-20.

[0233] In another embodiment, the present invention provides an antibody that specifically binds to an Omicron variant of SARS-CoV-2, wherein the antibody has a v region derived from IGHV1-69. It was surprisingly found that the antibody response to the Omicron variant infected with SARS-CoV-2 was biased towards antibodies having a heavy chain variable region derived from IGHV1-69. In one embodiment, wherein the antibody heavy chain is derived from IGHV1-69, the antibody of the present invention comprises CDRH1, CDRH2 and CDRH3 from Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34 and Omi38.

[0234] Antibody conjugates

[0235] The present invention also relates to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Conjugates of antibodies and cytotoxic agents can be made using various bifunctional protein coupling agents known in the art.

[0236] The antibodies of the invention may be conjugated to molecules that modulate or alter serum half-life. The antibodies of the invention may bind to albumin, for example, to modulate serum half-life. In one embodiment, the antibodies of the invention will also include a binding region specific for albumin. In another embodiment, the antibodies of the invention may include a peptide linker that is an albumin binding peptide. Examples of albumin binding peptides are included in WO 2015 / 197772 and WO 2007 / 106120, the entire contents of which are incorporated by reference.

[0237] Polynucleotides, vectors and host cells

[0238] The present invention also provides one or more isolated polynucleotides (e.g., DNA) encoding antibodies of the present invention. In one embodiment, polynucleotide sequences are present in more than one polynucleotide, but they can encode antibodies of the present invention together. For example, polynucleotides can encode the heavy chain and / or light chain variable region of antibodies of the present invention. Polynucleotides can encode complete heavy chains and / or light chains of antibodies of the present invention. Typically, a polynucleotide will encode each of the heavy and light chains.

[0239] The polynucleotides encoding the antibodies of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of the antibodies can be synthesized from the corresponding amino acid sequences as needed. The general methods, transfection methods and culture methods for constructing vectors are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology," 1999, FM Ausubel (ed.), Wiley Interscience, New York, and the Maniatis manual published by Cold Spring Harbor Press. The polynucleotides of the present invention can be provided in the form of an expression cassette, which includes a control sequence operably linked to an insertion sequence, thereby allowing the antibodies of the present invention to be expressed in vivo. Therefore, 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 generally provided in a vector (e.g., a plasmid or a recombinant viral vector). Therefore, 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 together. These vectors can be cloning vectors or expression vectors. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptides of the present invention. For example, polynucleotides, expression cassettes or vectors of the present invention are introduced into host cells by transfection. Therefore, the present invention also provides a host cell comprising one or more polynucleotides, expression cassettes or vectors of the present invention. The polynucleotides, expression cassettes or vectors of the present invention can be transiently or permanently introduced into host cells, thereby allowing antibodies to be expressed from one or more polynucleotides, expression cassettes or vectors. Such host cells include transient or preferably stable higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast) or prokaryotic cells (such as bacterial cells). Specific examples of cells include mammalian HEK293 (such as HEK293F, HEK293T, HEK293S or HEK Expi293F), CHO, HeLa, NS0 and COS cells or any other cell lines used herein (such as the cell lines used in the examples). Preferably, the selected cell line will be a cell line that is not only stable but also allows mature glycosylation.

[0240] The present invention also provides a process for producing an antibody of the present invention, comprising culturing a host cell containing one or more vectors of the present invention under conditions suitable for expressing the antibody from one or more polynucleotides of the present invention, and isolating the antibody from the culture.

[0241] Antibody Combination

[0242] The inventors have found that certain Table 3 antibodies are particularly effective when used in combination, and certain combinations of Table 3, Table 2, and Table 1 antibodies (e.g., to minimize loss of activity due to SARS-CoV-2 variants) maximize therapeutic efficacy and / or increase diagnostic power. Useful combinations include antibodies that do not cross-compete with each other and / or bind to non-overlapping epitopes.

[0243] Therefore, the present invention provides an antibody combination of the present invention, wherein each antibody is capable of binding to the spike protein of the coronavirus SARS-CoV-2, and wherein at least one antibody comprises at least three CDRs of any one of the 28 antibodies in Table 3.

[0244] The antibody combination of the invention can be used as a therapeutic cocktail. Therefore, the present invention also provides a pharmaceutical composition comprising the antibody combination of the invention, as further explained below.

[0245] The antibody combination of the present invention can be used for diagnosis. Therefore, the present invention also provides a diagnostic kit comprising the antibody combination of the present invention. A method for diagnosing a disease or complication associated with a coronavirus infection in a subject is also provided herein, as further explained below. A fully cross-neutralizing antibody (e.g., Omi03) can be used as a reference to confirm the presence and / or amount of any variant of interest (VoC) SARS-CoV-2 in a sample. Antibodies that bind to a limited number of VoCs can be used to confirm the presence and / or amount of the VoC in a sample. For example, if Omi03 shows binding to a sample, but Omi24 does not show binding to a sample of SARS-CoV-2, then the spike protein may be a spike protein of Delta VoC. This can be determined by any method known to the skilled person, such as via an immunoassay, such as an ELISA or immunochromatographic assay. Reduced binding can be determined by comparison with a reference and / or normalization to a reference and / or by comparison with a positive / negative control sample or data.

[0246] Pharmaceutical composition

[0247] The present invention provides a pharmaceutical composition comprising the antibody of the present invention. The composition may comprise a combination (such as two, three or four) of the antibodies of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0248] The compositions of the present invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0249] Suitable pharmaceutically acceptable carriers include aqueous carriers or diluents.

[0250] Examples of suitable aqueous carriers include water, buffered water, and saline.

[0251] Other suitable pharmaceutically acceptable carriers include ethanol, polyols (such as 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 (e.g., sugars), polyols (such as mannitol, sorbitol) or sodium chloride in the composition.

[0252] The therapeutic composition typically must be sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.

[0253] The pharmaceutical composition of the present invention may include additional therapeutic agents, such as antiviral agents. Antiviral agents can bind to coronaviruses and inhibit viral activity. Alternatively, antiviral agents may not bind directly to coronaviruses, but still affect viral activity / infectivity. The antiviral agent can be another anti-coronavirus antibody that binds to somewhere other than the spike protein on SARS-CoV-2. Examples of antiviral agents that can be used in the present invention include Remdesivir, Lopinavir, Ritonavir, APN01, and Favilavir.

[0254] The additional therapeutic agent may be an anti-inflammatory agent, such as a corticosteroid (eg, dexamethasone) or a nonsteroidal anti-inflammatory drug (eg, Tocilizumab).

[0255] The additional therapeutic agent may be an anti-coronavirus vaccine. The pharmaceutical composition may be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally. Also within the scope of the invention is a kit comprising the antibodies or other compositions of the invention and instructions for use. The kit may also contain one or more additional agents, such as additional therapeutic or preventive agents as discussed herein.

[0256] Methods and uses of the present invention

[0257] The invention also relates to the use of the antibodies, antibody combinations and pharmaceutical compositions described herein, for example in a method of treating the human or animal body by therapy or in a method of diagnosis. The method of treatment may be therapeutic or prophylactic.

[0258] For example, the present invention relates to a method for treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith (e.g., COVID-19). The method may comprise administering a therapeutically effective amount of an antibody, an antibody combination, or a drug combination of the present invention. The method may further comprise identifying the presence of a coronavirus or a fragment thereof (e.g., SARS-CoV-2) in a sample from a subject. The present invention also relates to an antibody, an antibody combination, or a pharmaceutical composition according to the present invention for use in a method for treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith (e.g., COVID-19).

[0259] The present invention also relates to a method for formulating a composition for treating coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith (e.g., COVID-19), wherein the method comprises mixing an antibody, antibody combination or pharmaceutical composition according to the present invention with an acceptable carrier to prepare the composition.

[0260] The present invention also relates to the use of an antibody, antibody combination or pharmaceutical composition according to the present invention for treating coronavirus (e.g., SARS-CoV-2) infection or a disease or complication associated therewith (e.g., COVID-19).

[0261] The present invention also relates to the use of an antibody, antibody combination or pharmaceutical composition according to the present invention for the manufacture of a medicament for treating or preventing coronavirus (e.g., SARS-CoV-2) infection or a disease or complication associated therewith (e.g., COVID-19).

[0262] The present 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.

[0263] SARS-CoV-2 strains may be the earliest identified ancestral strain ((hCoV-19 / Wuhan / WIV04 / 2019(WIV04); GISAID accession number: EPI_ISL_402124) and its variants. For example, SARS-CoV-2 strains may be 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), d The SARS-CoV-2 strain can be a member of lineage 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.

[0264] Relative to hCoV-19 / Wuhan / WIV04 / 2019 (WIV04) (GISAID accession number: EPI_ISL_402124), the SARS-CoV-2 strain may, for example, comprise one or more mutations in the spike protein. In other words, the SARS-CoV-2 strain may be a modified hCoV-19 / Wuhan / WIV04 / 2019 (WIV04) strain, for example, comprising one or more modifications in the spike protein.

[0265] The mutation may be a mutation (e.g., a substitution) observed in the Omicron strain of SARS-CoV-2. 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 treating, preventing, or diagnosing coronavirus infections caused by SARS-Cov-2 strains.

[0266] The methods and uses of the present invention may include inhibiting a disease state (such as COVID-19), for example, preventing its development; and / or alleviating a disease state (such as COVID-19), for example, causing regression of the disease state, until a desired endpoint is reached.

[0267] The methods and uses of the present invention may include improving or reducing the severity, duration or frequency (e.g., reducing pain or discomfort) of symptoms of a disease state (such as COVID-19), and such improvements may or may not directly affect the disease. Symptoms or complications may be fever, headache, fatigue, loss of appetite, myalgia, 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).

[0268] The methods and uses of the present invention can result in a reduction in the viral load of a coronavirus (e.g., SARS-CoV-2), for example, a reduction of ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90% or 100% compared to before treatment. Methods for determining viral load are well known in the art, such as infection assays.

[0269] The methods and uses of the present invention may include preventing coronavirus infection from occurring in a subject (eg, a human), particularly when the subject is susceptible to complications associated with coronavirus infection.

[0270] The present invention also relates to identifying subjects infected with coronaviruses (such as SARS-CoV-2). For example, the methods and uses of the present invention may involve identifying the presence of a coronavirus (e.g., SARS-CoV-2) or a protein or fragment thereof in a sample. The detection may be performed in vitro or in vivo. In certain embodiments, the present invention relates to population screening.

[0271] The present invention relates to identifying any SARS-CoV-2 strain, as described herein. The present invention may also relate to a method for identifying escape mutants of SARS-CoV-2, the method comprising contacting a sample with a combination of antibodies of the present invention and identifying whether each antibody binds to the virus. The term "escape mutant" refers to a variant of SARS-CoV-2 that contains non-silent mutations that may affect the efficacy of existing treatments for SARS-CoV-2 infection. Typically, the non-silent mutations are on an epitope recognized by a prior art antibody and / or an antibody described herein that specifically binds to an epitope of SARS-CoV-2, such as on the spike protein of SARS-CoV-2. If the antibody does not bind to the target, it may indicate that the target contains a mutation that may alter the efficacy of existing SARS-CoV-2 treatments.

[0272] The methods and uses of the invention may comprise contacting a sample with an antibody or antibody combination of the invention, and detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates that the subject is infected with SARS-CoV-2.

[0273] Methods for determining the presence of an antibody-antigen complex are known in the art. For example, in vitro detection techniques include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vivo techniques include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive marker, and its presence and location in the subject can be detected by standard imaging techniques. Detection techniques can provide qualitative or quantitative readings, depending on the assay used.

[0274] In general, the present invention relates to methods and uses for human subjects in need. However, non-human animals, such as rats, rabbits, sheep, pigs, cattle, cats or dogs are also contemplated. The subject may be at risk of exposure to coronavirus infection, such as a healthcare worker or a person who has come into contact with an infected individual. The subject may have visited or plans to visit a country where a coronavirus outbreak is known or suspected. The subject may also be at greater risk, such as an immunocompromised individual, such as an individual receiving immunosuppressive therapy or an individual suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS). The subject may be asymptomatic or before symptoms appear.

[0275] The subject may be in the early, middle or late stages of the disease.

[0276] Participants may have been in the hospital or community at the time of the first episode and / or in the hospital subsequently.

[0277] The subject can be male or female.

[0278] In certain embodiments, the subject is typically male. The subject may not be infected with a coronavirus, such as SARS-CoV-2. The subject may be susceptible to more severe symptoms or complications associated with a coronavirus infection. The methods or uses of the present invention may include a step of identifying whether a patient is at risk of developing more severe symptoms or complications associated with a coronavirus.

[0279] In embodiments of the invention involving prevention or treatment, the subject may or may not have been diagnosed with infection with a coronavirus, such as SARS-CoV-2.

[0280] The present invention relates to analyzing samples from subjects. Samples can be tissues, cells and biological fluids separated from subjects, as well as tissues, cells and body fluids present in subjects. Samples can be blood and a portion or component of blood, including serum, plasma or lymph. Typically, samples are from pharyngeal swabs, nasal swabs or saliva.

[0281] Antibody-antigen complex detection assays can be performed in situ, in which case the sample is a tissue section (fixed and / or frozen) of tissue obtained from a biopsy or resection of a subject.

[0282] In embodiments of the present invention, where antibody pharmaceutical 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.

[0283] The dosage of the antibody may vary depending on the age and size of the subject, as well as the disease, condition, and route of administration. The antibody may be administered at a dosage of about 0.1 mg / kg body weight to about 100 mg / kg body weight (such as at a dosage of about 5 mg / kg to about 10 mg / kg). The antibody may also be administered at a dosage of about 50 mg / kg, 10 mg / kg, or about 5 mg / kg body weight.

[0284] The combination of the present invention can be administered, for example, at a dosage of about 5 mg / kg to about 10 mg / kg of each antibody or at a dosage of about 10 mg / kg or about 5 mg / kg of each antibody. Alternatively, the combination can be administered at a total dosage of about 5 mg / kg (e.g., in a three-antibody combination, a dosage of 1.67 mg / kg of each antibody).

[0285] The antibodies or antibody combinations of the present invention can be administered in a multiple dose regimen. For example, a second or multiple subsequent doses can be administered after an initial dose. The second and subsequent doses can be spaced apart by appropriate time.

[0286] As discussed above, the antibodies of the invention are generally used in a single pharmaceutical composition / combination (co-formulation). However, the invention generally also includes the combined use of the antibodies of the invention in separate formulations / compositions. The invention also includes the combined use of the antibodies as described above with additional therapeutic agents.

[0287] The combined administration of two or more agents and / or antibodies can be achieved in a variety of different ways. In one embodiment, all components can be administered together in a single composition. In another embodiment, each component can be administered separately as part of a combination therapy.

[0288] For example, an antibody of the invention can be administered before, after, or simultaneously with another antibody of the invention or its binding fragment. Particularly useful combinations are described, for example, above.

[0289] For example, an antibody of the invention can be administered before, after, or simultaneously with an antiviral agent or an anti-inflammatory agent.

[0290] In embodiments of the present invention relating to the presence of a coronavirus (e.g., SARS-CoV-2) or a protein or fragment thereof in a detection sample, the antibody contains a detectable label. Methods for attaching labels to antibodies are known in the art, such as directly labeling antibodies by coupling (i.e., physically linking) a detectable substance to the antibody. Alternatively, the antibody may be indirectly labeled, such as by reacting 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 DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0291] The test may also include: (i) agents known to be useful for detecting coronaviruses (e.g., SARS-CoV-2) or proteins or fragments thereof, such as antibodies against other epitopes of the spike protein or other proteins of the coronavirus, such as anti-nucleocapsid antibodies; and / or (ii) agents known not to detect the presence of coronaviruses (e.g., SARS-CoV-2) or fragments thereof, i.e., providing a negative control.

[0292] In certain embodiments, the antibody is modified to have increased stability. Suitable modifications are explained above.

[0293] The present invention also includes a kit for detecting the presence of a coronavirus (e.g., SARS-CoV-2) in a sample. For example, the kit may include: a labeled antibody or a combination of labeled antibodies of the present invention; a device for determining the amount of a coronavirus (e.g., SARS-CoV-2) in a sample; and a device for comparing the amount of a coronavirus (e.g., SARS-CoV-2) in a sample with a standard. The labeled antibody or combination of labeled antibodies may be packaged in a suitable container. The kit may also include instructions for using the kit to detect a coronavirus (e.g., SARS-CoV-2) in a sample. The kit may also include other agents known to be useful for detecting the presence of coronaviruses, as discussed above.

[0294] For example, the antibody or antibody combination of the present invention is used for lateral flow test. Typically, a lateral flow test kit is a handheld device with an absorbent pad, which is based on a series of capillary beds, such as porous paper, microstructured polymers or sintered polymers. The test runs a liquid sample along the pad surface with reactive molecules showing visual positive or negative results. The test may also include the use of other agents known to be useful for detecting the presence of coronaviruses (e.g., SARS-CoV-2) or fragments thereof, as discussed above, such as anti-nucleocapsid antibodies.

[0295] other

[0296] It should be understood that the different applications of the disclosed antibody combinations or pharmaceutical compositions of the present invention can be adjusted according to the specific needs of the art. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments of the present invention and are not intended to be limiting. In addition, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes two or more "antibodies".

[0297] Furthermore, when "≥x" is mentioned herein, this means equal to or greater than x. When "≤x" is mentioned herein, this means less than or equal to x.

[0298] For the purposes of the present invention, in order to determine the percent identity of two sequences (such as two polynucleotides or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in the first sequence to optimally align with the second sequence). The nucleotides or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotide or amino acid at that position is identical. The percent identity between the 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 length of a 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 the reference sequence. In order to assess whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), a technician will align the length of SEQ ID NO: 3 and determine how many positions in the test sequence are identical to the positions of 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. The skilled artisan is aware of various computer programs that can be used to determine homology or identity between two sequences. For example, comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm (which has been incorporated into the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / The GAP program (obtained) was used and determined using the Blosum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0299] According to the IMGT numbering system (http: / / www.imgt.org; Lefranc MP, 1997, J, Immunol. Today, 18, 509), the CDRs of the heavy chain (CDRH) and light chain variable domains (CDRL) are located at residues 27-38 (CDR1), residues 56-65 (CDR2), and residues 105-117 (CDR3) of each chain. This numbering system is used in this specification unless otherwise indicated.

[0300] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0301] The following examples illustrate the invention.

[0302] Examples

[0303] Example 1. Generation of specific antibodies against early pandemic SARS-CoV-2 and Beta SARS-CoV-2 strains

[0304] The antibodies in Table 1 are related to a group of mAbs produced against the early pandemic strains of SARS-CoV-2. The antibodies in Table 2 are related to a group of mAbs produced against the Beta strain of SARS-CoV-2.

[0305] More details of these antibodies can be found in International Application Nos. PCT / GB2022 / 050306 and PCT / GB2022 / 050307. Further information on the generation and properties of these antibodies can be found in the following articles: 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.

[0306] Liu, Chang, et al. “The antibody response to SARS-CoV-2Beta underscores theantigenic distance to other variants.” Cell hostµbe (2021).

[0307] 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.

[0308] Dejnirattisai, Wanwisa et al. "Antibody evasion by the P.1 strain of SARS-CoV-2." Cell 184.11(2021):2939-2954.

[0309] Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16 (2021): 4220-4236.

[0310] Dejnirattisai, Wanwisa et al. “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell (2022).

[0311] Example 2. Generation of specific antibodies against the Omicron strain of SARS-CoV-2

[0312] Omicron BA.2 lineage

[0313] Omicron BA.2 was first reported in South Africa on November 17, 2021, similar to the time when Omicron BA.1 was reported. In many countries such as Denmark, India, and the United Kingdom, BA.2 has been increasing relative to BA.1 and now accounts for the majority of Omicron infections in Denmark, and there is increasing evidence that BA.2 is more transmissible than BA.1, but there is no evidence of increased disease severity.

[0314] BA.2 is related to BA.1, sharing 21 amino acid substitutions throughout S, but also with many differences. BA.1 has an additional 6 amino acid deletions, 3 insertions, and 9 substitutions compared to BA.2, and BA.2 has an additional 3 deletions and 7 substitutions compared to BA.1. In the RBD, BA.1 contains unique mutations S371L, G446S, and G496S, and in some isolates contains R346K (BA.1.1), while BA.2 carries S371F, T376A, D405N, and R408S. All of these residues have the potential to have different effects on antibody binding and can modulate neutralization, especially BA.1G446S, G496S, BA.2D405N, R408S, which are located at the edge of the ACE2 binding footprint, and for BA.1.1, the R346K change is close to the N343 glycan and can modulate the binding of potent antibodies to this region. BA.3 does not contain unique mutations relative to BA.1 and BA.2 and appears to be a fusion of the two, being BA.1-like at the N-terminus and converted to BA.2-like at the C-terminus by the mutation G496S.

[0315] Omicron lineages BA.4 and BA.5

[0316] In early April 2022, two new Omicron lineages were reported in Gauteng Province, South Africa, 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 Fig. 9 Compared to BA.2, BA.4 lacks residues 69 and 70 and contains 2 additional substitutions in the RBD: L452R and F486V. Finally, BA.4 lacks the Q493R change seen in BA.1 and BA.2, reverting to Q493 as in the Victoria / Wuhan strain.

[0317] Two additional mutations in the RBD are of greatest interest in terms of antibody escape: L452R is a chemically radical change and is one of a pair of changes in the Delta RBD (the other is T478K, which has been found in the Omicron lineage). Mutation F486L was found in sequences of SARS-CoV-2 isolated from mink early in the pandemic and is also an escape mutation site for several mAbs (Gobeil et al., 2021, “Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity.” Science 373, 6555). Change F486V in BA.4 / 5 also reduces a large portion of the hydrophobic side chain like F486L, but more significantly. Residues 452 and 486 are both close to the edge of the ACE2 interacting surface ( Fig. 9 B), and these two residues together with the back mutation to the ancestral sequence Q493 located within the ACE2 footprint have the potential to modulate ACE2 affinity and the neutralizing capacity of vaccines or naturally acquired sera. The L452R and F486V mutations are likely to result in more antibody escape, while the back mutation at 493 may reduce escape from early viral responses.

[0318] Omicron Lineage BA.2.75

[0319] In early May 2022, a new Omicron BA.2 sublineage named BA.2.75 was reported in India. It has spread to multiple countries, including the United Kingdom, the United States, Australia, Germany, and Canada. Compared with BA.2, BA.2.75 contains multiple mutations in the S protein, including four substitutions in the NTD (W152R, F157L, I210V, and G257S) and four substitutions in the RBD: D339H, G446S, N460K, and R493Q ( Fig.16). RBD mutations affect the main epitope of neutralizing antibodies and have the potential to modulate ACE2 binding. D339H represents a further evolution of the G339D mutation found in all previous Omicron variants, which has been found to impair the binding of certain “right-wing” antibodies belonging to the IGHV1-69 family (e.g., Beta-49 and -50); it also belongs to the binding footprint of certain class 3 antibodies such as S309 / sotrovimab (Dejnirattisai et al., 2022; “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell 185, 467-484e415). G446S was found in BA.1, BA.1.1, and BA.3, but not in BA.2 and other BA.2 subvariants, and can also impair the binding of certain class 3 antibodies (such as REGN10987 / imdevimab) that bind to the right shoulder (Dejnirattisai et al., 2022). The R493Q reversion mutation was also found in BA.4 / 5, which can make the virus more sensitive to neutralization by many class 1 and class 2 antibodies that bind to the neck / left shoulder. This reversion mutation can also increase affinity for ACE2 (see below).

[0320] N460K is a novel mutation not seen previously in the VoC or Omicron sublineages, but it was discovered after in vitro (yeast display) evolution of RBD-62 with ultra-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 [Nature Microbiology] 6, 1188-1198). In fact, N460K leads to a substantial increase in affinity for ACE2, second only to the effect of 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) that have been shown to potently neutralize all VoCs (Nutalai et al., 2022).

[0321] Using neutralization assays, it was shown that Delta infection alone provided no protection against BA.2.75 (no neutralization). Mutations in BA.2.75 resulted in reduced neutralization titers in vaccine sera compared to BA.2. Single BA.2.75 mutations can result in greater reductions in neutralization titers compared to the complete BA.2.75S sequence, but these mutations are balanced by the R393Q reversion mutation, which has been selected to increase affinity for ACE2 and increase the transmissibility of BA.2.75. It seems inevitable that further evolution of the Omicron lineage will occur, and there may be many possible trade-offs between antibody escape and ACE2 affinity that can and will be made, leading to successive waves of infection.

[0322] Newly emerged sublineages: BA.2, BA.4 and BA.5

[0323] Many lineages within the BA.2 and BA.5 branches are growing rapidly. The most striking is the large degree of convergent evolution, especially at antigenic RBD positions such as 346, 444, 452, 460, 486, 490, 493 and 494. These lineages include examples from the BA.4 / 5 branch (containing L452R, F486V and back mutation R493Q), such as BA.4.6 and BF.7 (R346T), BA.4.7 (R346S), BQ.1 (K444T, N460K) and B0.1.1 (R346T, K444T, N460K); from the BA.2.75 branch (containing G339H, G446S, N460K and back mutation R493Q), BA.2.75.2 (R346T and F486S and BA.2.75 mutations), BN.1 (also known as BA.2.75.5.1, with R346T, K356T, F490S and BA.2.75 mutations), BM.1.1.1 (also known as BA.2.75.3.1.1.1, with R346T, F486S, F490S and BA.2.75 mutations). There are several other examples of second generation BA.2 variants, such as BJ.1 (also known as BA.2.10.1.1; G339H, R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (G446S, F486P, S494P and R493Q reversion mutations), BS.1 (also known as BA.2.3.2.1; R346T, L452R, N460K, G476S and Q493R reversion mutations), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and Q493R reversion mutations) and finally BJ.1x BM.1.1.1 (also known as BA.2.75.3.1.1.1) recombinant XBB (which contains R346T, L368I, V445P, G446S, N460K, F486S, F490S and Q493R reversion mutations relative to BA.2).

[0324] Outside the RBD, the extent of convergent evolution is less, but still present. Many second-generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in the VoC, such as Δ~144 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).

[0325] Potent neutralizing antibodies isolated after Omicron infection

[0326] Five volunteers who had recovered from sequence-confirmed Omicron infection were recruited and sampled 10-14 days after symptom onset; all volunteers had received 2 doses of the Pfizer BioNtech vaccine prior to infection with Omicron. First, neutralization assays were performed against Omicron BA.1 and Victoria, an early pandemic SARS-CoV-2 isolate that contains only a single amino acid substitution (S247R) in the S NTD compared to the sequence of the ancestral strain used in all current vaccines. In all cases, the lesion reduction neutralization 50% titer (FRNT50) for Omicron was above 100, but at this early time point, the titers were substantially lower than those for Victoria ( Figure 1 A).

[0327] B cells from five donors were stained with full-length BA.1 trimer and single cells were sorted by FACS ( Figure 1 B). After degenerate RT-PCR, heavy and light chain sequences were assembled into expression vectors using the Gibson reaction and the products were transfected into 293T cells. Culture supernatants were screened for reactivity to full-length BA.1 or wild-type S (WT Wuhan) as well as BA.1 RBD and NTD. A total of 1,122 single cells were sorted and 545 mAbs were recovered.

[0328] All mAbs cross-reacted between WT and BA.1 S by ELISA, suggesting that they may be generated by memory B cells induced by vaccination. Compared with a previous panel of monoclonal antibodies generated from primary 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), a higher proportion of omicron-specific mAbs were reactive to RBD (56%) compared with early pandemic mAbs (21%, P < 0.0001) ( Figure 1 C) In addition, 129 out of 545 isolated mAbs bound BA.1 NTD.

[0329] Isolation of a potent Omicron mAb

[0330] All ELISA-positive mAbs were subjected to neutralization assays, and those showing the highest activity were selected for further study. The most potent 28 mAbs were selected for comprehensive characterization, all of which showed BA.1 FRNT50 titers <100 ng / ml. 27 of the 28 bound to the RBD (one, Omi-41, bound to the NTD) by ELISA, and none cross-reacted with the SARS-CoV-1 S protein.

[0331] Checking gene usage ( Figure 1 D, Table 17) shows that 9 of the 28 mAb species belong to the VH3-53 and related VH3-66 gene families. VH3-53 and VH3-66 have been repeatedly isolated in SARS-CoV-2 infection, they form a common antibody response and bind to sites on the neck of the RBD and act to block ACE2 binding. It was previously observed that many VH3-53 and VH3-66 mAbs lost activity against VoC containing the N501Y mutation, although some VH3-53 antibodies (mAb 222 and Beta-27) were completely resistant to the N501Y changes found in Alpha, Beta, and Gamma, but the activity against Omicron BA.1 or BA.2 was knocked down.

[0332] Approximately half of the gene families observed in potent early pandemic antibodies (Table 1) were also present in the Omicron group ( Figure 1 C), perhaps the most striking difference is that VH1-69 was not characterized in the earlier antibodies, but was found in 6 of 28 in the potent Omicron group (2, 24, 30, 31, 34, and 38), and we also found it in 2 Beta antibodies (Beta 49 and 50, which bind to a site close to the N343 glycan). Analysis of the Omicron mAbs showed much longer CDR3 sequences, suggesting a different binding mode than Beta 49, 50. In the Beta group of mAbs, the expansion of the common response was found to be mediated by VH 4-39 (which binds to the epitope around the 501Y mutation) (6 of 27 mAbs), most of which lost activity against BA.1, and notably, none of the current Omicron mAb group is encoded by VH4-39.

[0333] We found higher levels of somatic mutations in both the heavy and light chains of the mAb Omicron compared to the early pandemic panel (means 9.00, 6.00, and 4.55, 4.25 for VH and VL, respectively, in the early pandemic. These results are consistent with the evolution of increased affinity for Omicron via vaccine-induced somatic mutations in memory B cells.

[0334] Broad neutralization of VoC by Omicron mAb

[0335] Neutralization assays were performed against a panel of 28 potent mAbs against Victoria and all variants of interest, Alpha, Beta, Gamma, Delta, and Omicron BA.1 ( Figure 2A -C, Tables 13 to 16 and 18). The likely origin of all these antibodies from vaccine-induced memory B cells is evident because Victoria's FRNT50 titers were at the high end of all VoCs tested for each mAb in almost all cases ( Figure 2A -C, Tables 13 to 16 and 18). Among them, 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, respectively, and FRNT90 titers of 67, 42, 20, 18, and 35 ng / ml.

[0336] The data presented in Tables 13, 14 and 16 include some IC50 data obtained using pseudovirus constructs. The data in Table 18 include IC50 results obtained entirely from authentic virus constructs.

[0337] 17 of the 28 antibodies cross-reacted against all VoCs, with FRNT50 titers varying <10-fold between all viruses. Omi-06, 24, 30, 31, 34, and 41 showed reduced or absent activity against Delta (3 of the 6 belong to the VH1-69 family) and may have epitopes affecting the L452R Delta mutation (Delta shares T478K with BA.1). Omi-09 and 32 antibodies performed poorly against Beta and Gamma and may be sensitive to E484K found in Beta and Gamma, but tolerate the E484A change in Omicron (Omicron shares N501Y and K417N with Beta, while Gamma is N501Y, K417T). Finally, although 129 anti-NTD mAbs were isolated, only one of them, Omi-41, showed a FRNT50 titer <100 ng / ml. Omi-41 showed neutralizing activity against Victoria, Alpha, Beta, and Gamma, but not against Delta, likely due to the unique spectrum of NTD variations found in Delta.

[0338] Neutralization of BA.1 compared to BA.1.1, BA.2 and BA.3

[0339] Lentivirus-based reporters were constructed and pseudotyped with S gene sequences from Victoria, BA.1, BA.1.1, BA.2, and BA.3. Neutralization assays with Omicron mAbs were performed in Figure 2B As shown in Tables 14 and 18, most antibodies showed little difference in neutralization of BA.1, BA.1.1, BA.2, and BA.3. However, there were some notable exceptions; for Omi-8, 29, and 32, BA.2 neutralization was reduced by 38, 3, and 158 fold, respectively, compared to BA.1, while for Omi-6, 24, 34, and 35, BA.1.1 neutralization was reduced by 40.9, 10.8, 7.8, and 6.6 fold, respectively, compared to BA.1, and for Omi-39 and 40, this neutralization was knocked out. Neutralization of BA.3 by the Omi-mAbs was similar to that of BA.2, with the exception of Omi-06 and Omi-36, where BA.3 neutralization titers were significantly lower than BA.1 or BA.2. For some reason, the NTD-binding mAb Omi-41 did not neutralize Victoria in the pseudovirus system but neutralized the live virus, which was also found in the early pandemic mAb 159, which showed potent activity against the live virus but no activity against the pseudovirus.

[0340] Pseudovirus neutralization curves for the mAb panel isolated from early pandemic cases and the mAb panel isolated from Beta cases are shown in Figure 4A , B and Table 15 show that in most cases, the neutralization titers against BA.1, BA.1.1 and BA.2 were similar, but there were some differences. mAb40, 278 and 318 neutralized BA.2 more than BA.1, while 222, Beta 22, 29, 54, 55 and 56 neutralized BA.1 better than BA.2. At the same time, Beta-53, which tightly bound to the N343 glycan, showed reduced neutralization against BA.1.1.

[0341] Neutralization of antibodies being developed for clinical use.

[0342] Finally, mAbs that are being developed for clinical use (among which many differences were found) were used to test neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 strains ( Figure 2C, Tables 16 and 18). Interestingly, the activity of known antibody A (REGN10987) was partially restored against BA.2, but was still 308-fold reduced compared to Victoria, the activity of known antibody D (AZD1061) was almost completely restored against BA.2, while known antibody D (AZD8895) was 5.4-fold reduced against BA.2 relative to BA.1, and the combination of known antibodies D (AZD8895) and E was only 8-fold reduced compared to Victoria. The activity of known antibody K (S309) was 6.8-fold reduced against BA.2 compared to BA.1, and finally the activity of known antibody G (ADG20) was completely lost against BA.2.

[0343] In summary, neutralization by most Omicron mAbs was not affected by differences between BA.1, BA.1.1, BA.2, or BA.3 mutations. However, some mAbs did show differences, notably known antibody A (REGN 10987) and known antibody C (AZD1 061) neutralized BA.2 more readily than BA.1, and known antibody K (S309) showed reduced neutralization of BA.2, which may prompt sublineage sorting prior to use. Structural explanations for the differences between neutralization of BA.1, BA.1.1, BA.2, and BA.3 are discussed below.

[0344] Neutralization of BA.1, BA.1.1, BA.2 and BA3 by immune sera

[0345] To determine whether the difference in transmissibility between BA.1 and BA.2 might be due to differential neutralization, and also to determine whether BA.2 might be able to evade the BA.1 antibody response, neutralization assays were performed using sera from various sources. First, neutralization assays were performed for Victoria, BA.1, BA.1.1, BA.2, and BA.3 using sera collected from vaccine recipients who received either the Oxford / AstraZeneca AZD1222 (n=41) or the Pfizer / BioNTech BNT162b2 (n=20) vaccines. Figure 3 A, B).

[0346] For AZD1222, samples were collected 4 weeks after the second and third doses of the vaccine. After the third dose of AZD1222, the difference in pseudovirus neutralization was small but significant, with titers to BA.2 reduced relative to BA.1 (1.17 times, p=0.0019), and titers to BA.1.1 reduced relative to BA.1 (1.29 times, p=0.0086). For BNT1 62b2, 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 vaccine dose, the titers for BA.1, BA.2, and BA.1.1 were similar, with no significant differences between them. Next, the neutralization of sera collected from cases infected with Omicron was determined. Early samples (n=12) were collected <14 days (median 13 days) after the onset of symptoms, and late samples (n=17) were collected >21 days (median 38 days) after the onset of symptoms. All cases had received at least 2 doses of vaccine, and many late-convalescent cases received a third dose of vaccine after Omicron infection. Neutralization of Victoria, Alpha, Beta, Gamma, Delta, and Omicron was tested using a live virus neutralization assay ( Figure 3 C). At early time points, all vaccinated cases had high titers to Victoria, with a geometric mean FRNT50 approaching 1 / 3000, and showed broad neutralization to VoC, with FRNT50 > 1 / 1000 for all viruses except Omicron (FRNT50 = 558). At later time points, titers to Victoria did not change, while titers to VoC and Omicron increased (3-fold, p = 0.0123). Paired comparisons of early and late samples from the same individuals confirmed a broad enhancement of the response to Omicron infection ( Figure 5 A).

[0347] Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 was determined by pseudovirus neutralization. BA.1 neutralization titers were higher at later time points. However, all sera were obtained from BA.1 infected cases, and the neutralization titer of BA.2 was slightly but significantly reduced relative to BA.1 (1.7-fold and 1.5-fold at <14 days and >21 days, respectively, p=0.0034 and 0.0067), the titer of BA.1.1 was not significantly reduced relative to BA.1, and at >21 days, the titer against BA.3 was reduced by 1.7-fold relative to BA.1 (p=0.0012) ( Figure 3 D, 5B).

[0348] In summary, after three doses of vaccine, especially BNT162b2, good neutralizing titers of antibodies against Omicron BA.1BA.1.1, BA.2, and BA.3 were induced, with only minor differences between titers against BA.1BA.1.1, BA.2, and BA.3. This may indicate that the increased transmissibility of BA.2 is not due to increased vaccine escape. After breakthrough Omicron infection, broad antibody responses to variants of concern were enhanced in previously vaccinated individuals, with strong responses to Omicron. Since there were only minor differences in neutralization between BA.1 and BA.2, BA.2 superinfection is unlikely in BA.1-exposed and vaccinated cases, at least in the short term.

[0349] Neutralization of BA.4 compared to BA.1, BA.1.1, BA.2 and BA.3

[0350] Neutralization of BA.4 / 5 was also evaluated compared to Omicron sublineage BA.1, BA.1.1, BA.2, BA.3, and early pandemic Victoria strains. BA.4 / 5 has been shown to have a more extreme antibody escape phenotype than BA.1 and BA.2, and neutralization titers of sera from triple-vaccinated donors were reduced by approximately 2-3 times compared to neutralization of BA.1 and BA.2. In addition, sera from breakthrough BA.1 infections in vaccine recipients showed approximately 2-3 times reduced neutralization titers against BA.4 / 5 compared to BA.1 and BA.2. This suggests that currently approved vaccines and mAbs may be less effective in preventing transmission of BA.4 / 5. Therefore, new mAbs and combinations may be needed to fill gaps to protect extremely vulnerable populations and those who are unable to mount an adequate vaccine response.

[0351] Neutralization of BA.4 by vaccine sera

[0352] A panel of pseudotyped lentiviruses expressing the S gene from the Omicron sublineages BA.1, BA.1.1, BA.2, BA.3, and BA.4 / 5 was constructed (Di Genova et al., 2020, “Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes”. Figshare preprint[Figshare preprint].), as well as an early pandemic ancestral related strain Victoria used as a control.

[0353] Using the Oxford-AstraZeneca vaccine ADZ1222 (n=41) after the third dose (Flaxman et al., 2021, "Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx 1nCoV-19 in the UK: a substudy of two randomised controlled trials (COV00 1and COV002)". Lancet [Lancet / ] 398, 981-990) or the Pfizer-BioNTech vaccine BNT162b2 (Cele et al., 2021, "Omicron extensively but incompletely escapes Pfizer Neutralization assay was performed with serum obtained 28 days after BNT162b2 neutralization [Omicron extensively but not completely evades Pfizer BNT162b2 neutralization]". Nature [Nature] 602, 654-666) (n = 20) Figure 7 A, B). For AZD1222, neutralization titers of BA.4 were reduced by 2.1-fold compared to BA.1 (p=0.0001) and by 1.8-fold compared to BA.2 (p=0.0001). For BNT1 62b2, neutralization titers were reduced by 3.2-fold (p=0.0001) and 3.1-fold (p=0.0001) compared to BA.1 and BA.2, respectively. These titer reductions have the potential to reduce vaccine effectiveness, especially at longer time points, as antibody titers naturally wane.

[0354] Neutralization of BA.4 / 5 by sera from breakthrough BA.1 infection

[0355] Vaccinated volunteers who suffered breakthrough Omicron infection were recruited at the onset of the Omicron outbreak. Samples were first collected ≤14 days (median 13 days) after symptom onset, while later samples were collected ≥21 days (median 38 days) after symptom onset (n=16). Pseudovirus neutralization assays were performed on a panel of pseudoviruses representing variants of concern and Omicron sublineages ( Figure 7 C, D).

[0356] BA.1 infection after vaccination resulted in a broad neutralizing 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 reduced by 1.9-fold (p=0.0001) and 1.5-fold (p=0.0015) compared to BA.1 and BA.2, respectively, at early time points. Before later time points, BA.4 / 5 titers were reduced by 3.4-fold (p=0.0001) and 2-fold (p=0.0017) compared to BA.1 and BA.2, respectively.

[0357] Therefore, BA.4 / 5 showed some degree of immune escape from the vaccine / BA.1 response when compared to BA.1 and BA.2. These samples were all collected fairly close to the time of infection, meaning that further attenuation in the intervening months may have left individuals vulnerable to reinfection with BA.4 / 5.

[0358] BA.4 / 5 escapes monoclonal antibodies

[0359] Sensitivity to L452R: It has been previously reported that Omi-24, 30, 31, 34, and 41 showed complete knockout of neutralizing activity against Delta, while Omi-06 showed severe knockdown of activity (Nutalai et al., 2022). Since BA.1 and BA.2 have only one of the two Delta RBD mutations (T478K), and BA.4 / 5 also have L452R, all five L452-directed mAbs were expected to knockout on BA.4 / 5. This was indeed observed ( Figure 8 A, Table 20). Omi-41 was also unable to neutralize, which was attributed to differences in mutations in the NTD ( Fig. 9 A).

[0360] To confirm that the observed neutralization was directly due to altered RBD interactions, the binding of the selected antibodies to BA.4 / 5 and BA.2 RBDs was also analyzed by surface plasmon resonance (SPR) ( Fig.10 , 15 Omi-31 was chosen as a representative of the L452R-sensitive antibody group and, as expected, binding was severely affected ( Fig.10 A).

[0361] Since detailed information on the interaction of several omicron-reactive antibodies with the RBD is available, the BA.4 / 5RBD mutations were modeled in the context of the known structures of the omicron Fab in complex with the BA.1 or Delta RBD (Dejnirattisai et al., 2022, “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.” Cell 185, 467-484e415; Nutalai et al., 2022) Fig.11 ). Omi-31 complex in Fig.11 This is shown in A and shows that L452 fits neatly into a hydrophobic pocket that cannot accommodate the larger positively charged arginine in BA.4 / 5 and Delta.

[0362] L452R enhances binding: Omi-32 enhances neutralization of BA.4 / 5 by 77-fold compared to BA.2. Kinetic analysis of Fab binding to RBD suggests that this is primarily achieved by increasing the binding rate by 5-fold ( Fig.10 B, C). This is mainly explained by the favorable interaction of the arginine at 452 with residue 99 of the heavy chain (HC) CDR3 to form a salt bridge ( Fig.11 B), perhaps aided by the removal of a slightly unfavorable charge interaction at residue 493. These electrostatic changes have the potential to increase the on-rate by exerting electrostatic control over the incoming antibody.

[0363] Sensitivity to F486V: Extending the logic used to understand Delta sensitivity, the remaining antibodies affected by BA.4 / 5>BA.2 but retaining activity to Delta are likely sensitive to the F486V change, namely Omi-02, 09, 12, 23, 25, 26, and 29. Binding sensitivity was confirmed by SPR analysis of Omi-12 ( Fig.10 D, E), showing an almost 1,000-fold reduction in affinity. The Omi-25 complex provides an example of the structural basis of sensitivity ( Fig.11 C), showing that the phenylalanine side chain acts as a binding hotspot, positioned in a hydrophobic cavity, thereby engaging in a favorable ring-like stacking interaction with Y106 of HC CDR3.

[0364] Activity of commercial antibodies against BA.4 and BA.5

[0365] A panel of antibodies developed for therapeutic / prophylactic use was tested against BA.4 / 5 ( Figure 8 B, Table 21). Many of these antibodies have severely reduced or knocked out activity against BA.1, BA.1.1, or BA.2. For AstraZeneca AZD1061, activity against BA.4 / 5 was similar to BA.2 (reduced <2-fold), while for AZD8895, residual activity against BA.2 was knocked out. The activity of the combination of two antibodies 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 reduced 8.1-fold compared to 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 reduced on BA.4 / 5, and similarly, residual BA.1 neutralizing activity was knocked out against ADG20 on BA.4 / 5 (Yuan et al., 2022, “A broad and potent neutralization epitope in SARS-related coronaviruses”. bioRxiv. https: / / doi.org / 10.1101 / 2022.03.13.484037). For 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), the activity against BA.4 / 5 was reduced 1.6-fold compared to BA.2.

[0366] These effects can be rationalized by reference to the way the antibody interacts with the RBD, as in the case of AZD8895 (a mAb for the IGHV1-58 genotype, Fig.11 E), F486 forms a hydrophobic interaction hotspot that would be abolished by mutation to the much smaller valine side chain. The antibody residues involved in the interaction with F486 are highly conserved among mAbs of this genotype, including Omi-12, 253, and Beta-47 (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, 2183-2200e2122; Liu et al., 2021, “The Beta mAb response underscores the antigenic distance to other SARS-CoV-2 variants”. Cell, Host and Microbe [Cell Host and Microbe] 30, 53-68), thus explaining the severe impact of the F486V mutation on the neutralization of these mAbs ( Figure 8 A, 13).

[0367] Neutralization of BA.2.75 by vaccine serum

[0368] A set of pseudotyped lentiviruses expressing the S gene from the Omicron sublineages BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, BA.2.75 were constructed as above (Di Genova et al., 2020), and Victoria (an early pandemic ancestor-related strain) was used as a control. D339H, G446S, N460K and R493Q were also included as single mutations in the BA.2 background. Using the Oxford-AstraZeneca vaccine AZD1222 (n=41) (Flaxman et al., 2021 "Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx 1nCoV-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 Neutralization assays were performed with serum obtained 28 days after BNT162b2 neutralization [Omicron extensively but not completely evades Pfizer BNT162b2 neutralization]. Nature [Nature] 602, 654-666e) Fig.17 For AZD1222, neutralization of BA.2.75 was reduced 1.2-fold compared to BA.2 (p=0.0182) and 1.1-fold compared to BA.2.12.1 (p=0.0065), but increased 1.5-fold compared to BA.4 / 5 (p<0.0001) ( Fig.17 B). Overall, the neutralization titers of vaccine sera were reduced for BA.2.75 compared to BA.2, but not to the levels seen for BA.4 / 5.

[0369] Neutralization of BA.2.75 by sera from vaccine breakthrough BA.1 or BA.2 infections

[0370] Breakthrough BA.1 serum samples were collected from vaccinated volunteers ≥28 days after symptom onset (median 38 days; n=16). Pseudovirus neutralization assays were performed on the pseudovirus panel described above ( Fig.17C). The neutralization titer of BA.2.75 was similar to that of BA.2 and was 1.4-fold (p=0.0052) and 2.0-fold (p=0.0001) higher than that of BA.2.12.1 and BA.4 / 5, respectively, suggesting that BA.2.75 may be less likely than BA.2.12.1 or BA.4 / 5 to cause reinfection in individuals with breakthrough infection with BA.1.

[0371] Breakthrough BA.2 serum samples were collected from vaccinated volunteers ≥12 days after symptom onset (median 29 days; n = 23). Pseudovirus neutralization assays were performed on the pseudovirus panels Victoria, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75 ( Fig.17 D). Here, the neutralization titer for BA.2.75 was significantly lower than that for BA.2 (1.4-fold; P = 0.0021), similar to that for BA.2.12.1, but still higher than that for BA.4 / 5 (1.4-fold; P = 0.0123). In summary, BA.2.75 showed a certain degree of escape from the humoral response induced by BA.2 breakthrough infection but not BA.1 infection.

[0372] Single BA.2.75 mutations have different effects on neutralization

[0373] To understand the impact of individual mutations in BA.2.75RBD, these mutations were introduced individually into the pseudovirus BA.2 background and their neutralization was determined using triple-vaccinated Pfizer BNT1 62b2 sera ( Fig.17 E). Three of the four single mutation variants of BA.2 reduced the neutralization titer against BA.2, with N460K reducing the most (3.1-fold, p < 0.0001), followed by D339H (1.3-fold, p = 0.0006), and then G446S (1.2-fold, p = 0.2312), whereas the R493Q reversion increased the neutralization titer by 1.5-fold (p < 0.0001). Q493 is present in all vaccines, explaining the increased activity of vaccine sera against this reversion.

[0374] BA.2.75 escapes monoclonal antibodies

[0375] To dissect how BA.2.75 might affect neutralizing antibody activity, a pseudovirus assay was used to test a recently reported panel of potent human mAbs generated from an Omicron breakthrough infection case (BA.1 IC50 titer <0.1 μg / ml) (Nutalai et al., 2022)( Fig.19A, Table 22). Of the 27 RBD-specific mAbs, those belonging to the IGHV3-53 / 66 family were the most severely affected. Three (Omi-16, Omi-29, and Omi-36) showed complete knockout of BA.2.75 neutralization; the other four (Omi-18, Omi-20, Omi-27, and Omi-28) showed a >5-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 RBD / IGHV-3 / 66 complex structure ( Fig.21 B)(Dejnirattisai et al., 2021, Liu et al., 2021, Nutalai et al., 2022).

[0376] Like BA.2 and BA.4 / 5, BA.2.75 was not neutralized by the anti-NTD mAb Omi-41 and it only interacted with the NTDs of BA.1, BA.1.1, and BA03.

[0377] Omi mAb was also tested against pseudoviruses encoding single point mutations in the BA.2 RBD described above ( Fig.23 , Table 24). The VH3-53 / 66 mAb that lost neutralization for BA.2.75 was also affected by the N460K mutation, confirming the prediction that this residue is critical for binding to many members of this common gene family. Interestingly, the BA.2+N460K mutation alone showed 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 for BA.2+N460K, but only 2-fold for BA.2.75; Omi-17 (IGHV3-66) was completely knocked out on BA.2+N460K, but only 4-fold for BA.2.75; and Omi-33 (IGHV3-33) was reduced 7-fold for BA.2+N460K, but no changes were observed for BA.2.75. Therefore, other mutations in BA.2.75 may mitigate the impact of the N460K mutation, especially the R4930 mutation.

[0378] Interestingly, BA.2.75 was more sensitive to Omi-32 (IGHV-3-33) than BA.2, with an 8-fold increase in neutralization titers. The enhanced activity of Omi-32 may be due to a stronger interaction of the antibody with the RBD via the G446S mutation ( Fig.19 A. Table 22).

[0379] To confirm that the observed changes in neutralization activity were associated with altered RBD interactions, the binding of the selected antibodies to BA.2.75 and BA.2 RBD was analyzed by surface plasmon resonance (SPR) ( Fig.24). Binding of Omi-29 (IGHV3-53) and Omi-36 (IGHV3-66) to BA.2.75 was severely impaired, and Omi-18 and Omi-20 showed an 8-fold decrease compared to BA.2. On the other hand, a 2-fold increase in binding affinity of Omi-32 to BA.2.75 was observed compared to BA.2, which is consistent with the observed enhancement in neutralization titers.

[0380] Evasion of commercial monoclonal antibodies against BA.2.75

[0381] A panel of mAbs that have been developed as therapeutic agents against BA.2.75 was evaluated ( Fig.19 B, Table 23). Neutralization between BA.2.75 and BA.2 was generally similar; however, except for 6 of the 12 mAbs that completely lost neutralizing activity against BA.2 (REGN10933, ADG10, ADG20, ADG30, Ly-CoV555, Ly-CoV16), REG10987 had residual activity against BA.2 (Weinreich et al., 2021, “REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19.” N Engl J Med [New England Journal of Medicine]384, 238-251) was further knocked out against BA.2.75 due to the G446S mutation (Dejnirattisai et al., 2022). For AstraZeneca AZD1061, the activity against BA.2.75 was similar to that against BA.2 (reduced by <3 times); while the titer of AZD8895 was restored from 1.333 μg / ml for BA.2 to 0.008 μg / ml for BA.2.75, an increase of 167 times in activity. Therefore, 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 lower) against BA.2.75 and BA.2. These results can be explained by the structure of the ternary complex of the ancestral SARS-CoV-2RBD / AZD1061 / AZD8895 (Dong et al., 2021). G446 is in contact with CDR-L2 Y55 and W56 of AZD1061, and the G446S mutation will cause steric conflict ( Fig.21 D, E). Although the CDR-H2 of AZD8895 is located above Q493 of RBD and forms a hydrogen bond with it, the arginine at 493 will seriously clash with the CDR-H2 of mAb ( Fig.21F, G). The S309 activity of BA.2.75 (Sun and Ho, 2020, “Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic.” Antib Ther 3, 246-256) was increased 3-fold compared to BA.2, indicating that the D339H mutation in BA.2.75 reduced the effect of the previous 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 that fully retains neutralization against all Omicron sublineages.

[0382] BA.2, BA.4, and BA.5 sublineages escape monoclonal antibodies

[0383] Decreased mAb activity was also observed in the novel 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). Fig.34 ), with XBB producing the most extreme escape. The activity of all nine IGHV3-53 / 66 mAbs was reduced >100-fold, with 5 / 9 having their activity completely abrogated by BA.2.75.2. Only one mAb, Omi-42, was unaffected by all variants. Omi-42 is unusual in that it binds to the posterior portion of the left shoulder of the RBD (Nuta1ai et al., 2022), a region that has not been targeted for mutation by this group of emerging BA.2 variants, perhaps because antibodies binding in this region are relatively rare.

[0384] Further data can be found in the following references: Nutalai et al. (2022) “Potent cross-reactive antibodies following Omicron breakthrough in vaccinees,” Cell 185(12), 2116-2131; Huo et al. (2022) “Humoral responses against SARS-CoV-2 Omicron BA.2.11, BA.2.12.1andBA.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 A delicate balance between the affinity of BA.2.75]” Cell Reports, 42(1).2023.

[0385] Neutralization of BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 by vaccine sera

[0386] The receptor binding abilities of BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 were also evaluated. A high-resolution crystal structure of BA.2.12.1 RBD was generated, showing the different sensitivities of the new BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 to serum samples and monoclonal antibodies (mAbs) compared to BA.2.

[0387] BA.2.13 is expected to be a relatively modest change given the physicochemical properties of the side chain at residue 452; the L to M change would increase the size of the side chain, but it would still be hydrophobic. The L to Q change in BA.2.12.1 would introduce some polar character, while BA.2.11 would be the most radical, with the L to R change introducing a large basic amino acid.

[0388] Neutralization of BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 by vaccine sera

[0389] To evaluate the sensitivity of BA.2 subvariants to neutralization by immune sera, a neutralization assay was performed on pseudotyped lentiviruses expressing the spike genes of BA.2.11, BA.2.12, and BA.2.13 using a series of serum samples.

[0390] First, the neutralization of sera collected 4 weeks after the third dose of the Oxford-AstraZeneca vaccine AZD1222 (n=41) or the Pfizer-BioNTech vaccine BNT162b2 (n=18) was observed. No significant loss of neutralization titers was observed compared to BA.2. In fact, BA.2.13 showed a significant increase in AZD1222 vaccine recipients (1.6-fold, p<0.0001) ( Fig. 27 a, b). This contrasts with a recent report (Cao, Y. et al., "BA.2.12.1, BA.4and BA.5 escape antibodies elicited by Omicron infection". Nature, 2022), in which sera collected from three-dose vaccine recipients (4 weeks after the third dose of the inactivated vaccine CoronaVac or the ZF2001 booster dose after two doses of CoronaVac) showed significantly reduced neutralizing titers for both BA.2.12.1 and BA.2.13 (BA.2.11 was not tested).

[0391] Neutralization of BA.2 subvariants BA.2.11, BA.2.12, and BA.2.13 by sera from vaccine breakthrough BA.1 or BA.2 infections

[0392] Next, neutralization of serum samples collected from vaccine recipients infected with BA.1 was examined. Samples (n=14) were collected ≥28 days (median 38 days) after symptom onset; all recovered individuals had received at least 2 doses of vaccine, and 3 received a third dose of vaccine after infection with Omicron. Neutralization titers were significantly reduced compared with BA.2, with BA.2.11 having the largest reduction (1.6-fold, P=0.0067), followed by BA.2.12.1 (1.4-fold, P=0.0085) and BA.2.13 (1.2-fold, P=0.0085) ( Fig. 27c). In summary, these observations suggest that, compared with BA.2, its subvariants did not show stronger humoral immune escape in individuals vaccinated with three doses of AZD1222 or BNT162b2. However, for vaccine recipients who suffered BA.1 breakthrough infection, the BA.2 variant was more able to evade the humoral response, regardless of the type of vaccine they had received, despite inducing a broad neutralizing antibody response with high titers against all variants of concern (Nutalai, R. et al., "Potent cross-reactive antibodies following Omicron breakthrough in vaccines". Cell, 2022. 185(12): pp. 2116-2131, e18). This may indicate that the selection pressures for BA.2 and its subvariants are different in the high background of breakthrough infection. Since antibody titers naturally weaken at longer time points, people who suffer BA.1 breakthrough infection are expected to be more susceptible to reinfection with the BA.2 subvariant.

[0393] To further elucidate the different responses between BA.2 and its subvariants, a panel of potent human monoclonal antibodies (mAbs) generated from a BA.1 breakthrough infection case was subjected to pseudovirus assays (Nutalai et al., 2022)( Fig.29 ). Consistent with the structural observations and neutralization results, the neutralization titer of BA.2.11 was observed to be the most reduced, followed by BA.2.12.1 and BA.2.13. For 5 of the 27 mAbs (Omi-06, Omi-24, Omi-30, Omi-31, and Omi-34), the neutralization of BA.2.11 was completely knocked out. The neutralizing activity of the same group of mAbs against BA.2.12.1 was also reduced to varying degrees, while the situation for BA.2.13 remained basically unchanged. Among them, Omi-06 belongs to the IGVH4-4 family, while the other four mAbs belong to the IGVH1-69 family. In fact, previous structural studies predicted that Omi-06 and Omi-31 were sensitive to the L452R mutation in Delta (Nutalai et al., 2022). To confirm that the observed differential neutralization was directly attributable to changes in RBD binding, surface plasmon resonance (SPR) was used to compare the binding behavior of BA.2 and BA.2.12.1 RBDs, using Omi-06 and Omi-31 as examples. As expected, the affinity decreased, with BA.2.12.1 RBD binding 15-fold weaker than BA.2 for Omi-06, and strikingly, BA.2.12.1 RBD binding to Omi-31 was ~1300-fold weaker ( Fig.30 ).

[0394] The 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, they do not appear to achieve greater humoral immune escape in healthy vaccine recipients who have received three doses of the Oxford-AstraZeneca or Pfizer-BioNTech BNT162b2 vaccines compared to BA.2. This result is different from that in vaccine recipients who have received three doses of the CoronaVac vaccine, in whom neutralizing titers were significantly reduced (Cao, Y. et al., BA.2.12.1, BA.4and BA.5 escape antibodies elicited by Omicron infection [BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection]. Nature [Nature], 2022). However, in vaccine recipients who experienced a breakthrough BA.1 infection, a significant reduction in neutralization titers was observed regardless of the type of vaccine received, perhaps in part due to partial or complete knockout of the neutralizing activity of antibodies belonging to the IGVH1-69 family, many of which are sensitive to mutations at leucine 452 of the spike RBD. This suggests that evolving Omicron sublineages are able to evade the humoral immune response elicited by BA.1, suggesting that the BA.1 spike virus or RBD may not be a better immunogen than the ancestral strain for the development of next-generation SARS-CoV-2 vaccines.

[0395] BA.2.12.1 Crystal structure

[0396] The crystal structure of BA.2.12.1 RBD was determined at 2.38 Å as a ternary complex with the neutralizing Fab and nanobody ( Fig. 27 hm), indicating that the structural differences are essentially limited to the side chain of residue 452.

[0397] Neutralization of BA.2.75.2 by mAbs produced after BA.1 infection

[0398] Neutralization of BA.2.75.2 by a panel of mAbs made after BA.1 infection was explored (Nutalai et al., 2022). A reduction in mAb activity against BA.2.75.2 was observed (Table 32a). The activity of all 9 IGVH3-53 / 66 mAbs was reduced by >100-fold, with 4 / 9 of them completely knocked out by BA.2.75.2. Only one mAb, Omi-42, was unaffected by all variants and showed neutralization against the BA.4+14 mutation with an IC50 of 11 ng / ml. Omi-42 is unusual in that it binds to the posterior portion of the left shoulder of the RBD (Nutalai et al., 2022), a region that has not been targeted for mutation, perhaps because antibodies that bind in this region are relatively rare.

[0399] A panel of mAbs that have been developed for clinical use was also tested (Dong et al., 2021; Sun and Ho, 2020; Weinreich et al., 2021; Yuan et al., 2022). Many of these mAbs were severely affected by many variants. With the exception of S309, the activity of all mAbs was knocked out by one or more variants, including Ly-CoV1404 (Westendorf et al., 2022) (see Table 32b).

[0400] Pfizer BNT162b2 vaccine serum neutralization titers for BA.2.75.2 and BA.2.3.20.

[0401] Neutralization of sera collected 28 days after the third dose of Pfizer BNT1 62b2 vaccine (Polack et al., 2020) and the characteristics of these subjects in cases infected with BA.1, BA.2 or BA.4 / 5 are described in the methods.

[0402] Using serum obtained 28 days after BNT162b, the infectivity titer of BA.2.75.2 showed a substantial reduction compared to BA.2 and BA.4, and was the lowest of all tested variants compared to the ancestral strain Victoria. The titer of BA.2.75.2 was reduced compared to BA.2.75 (which showed only a modest reduction compared to BA.2). The titer of BA.2.3.20 was also substantially reduced.

[0403] Using sera obtained after infection with BA.1, BA.2, and BA.4 / 5, BA.2.75.2 and BA.2.3.20 had similarly large reductions in neutralization titers compared to BA.4 / 5. Neutralization of the aforementioned BA.4+14RBD mutation was also reduced compared to BA.2 and BA.4 / 5, but not much more than BA.2.75.2, suggesting that the mutation in BA.2.75.2 has a dominant effect.

[0404] Neutralization of BA4.6 by sera from vaccine breakthrough BA.1 or BA.2 infections

[0405] Here, we investigated neutralization of BA.4.6 using Pfizer-BioNTech vaccine sera, BA.1, BA.2, and BA.4 / 5 breakthrough immune sera, and multiple panels of monoclonal antibodies. Notably, we demonstrated further antibody evasion of BA.4.6, providing guidance for vaccine design and the use of therapeutic monoclonal drugs.

[0406] To evaluate the antibody evasion ability of BA.4.6, we constructed 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, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes [SARS-CoV-2 lentiviral pseudotype production, titration, neutralization and storage]. figshare, 2020), as well as an early pandemic ancestral related strain Victoria used as a control. First, the neutralization of sera collected 4 weeks after the third dose of the Pfizer-BioNTech vaccine BNT1 62b2 (n=22) was examined. Compared with BA.4 / 5, the neutralization titer of BNT162b2 sera against BA.4.6 was reduced by 2-fold (p<0.0001) ( Fig.28 a).

[0407] Neutralization was determined in serum samples collected from vaccinees infected with BA.1. Samples (n=16) were collected ≥28 days after symptom onset. BA.2 or BA.4 / 5 samples (n=23) were collected ≥12 days after symptom onset. Samples (n=11; all but one vaccinee) were collected >23 days after symptom onset ( Fig.28 bd). Neutralization titers of BA.4.6 were significantly reduced in both breakthrough BA.1 (1.5-fold; P = 0.0006) and BA.2 (1.2-fold; P = 0.0384) serum samples compared to BA.4 / 5. Notably, BA.4.6 was able to effectively escape neutralization in serum samples from BA.1 breakthrough infections, showing a substantial reduction in titer 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). A small non-significant increase in neutralization titers against BA.4.6 was observed in the BA.4 / 5 breakthrough cohort compared to BA.4 / 5.

[0408] To further characterize the antigenic escape properties of BA.4.6, a panel of potent human mAbs generated from BA.1 breakthrough recoveries was subjected to pseudovirus assays (Nutalai et al., 2022)( Fig.28e). Overall, neutralization 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 against 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 attenuated against BA.4.6. The loss of activity of Omi-32 can be explained by the disruption of the interaction between H1 and R346, as previously illustrated by structural analysis (Nutalai et al., 2022).

[0409] Neutralization of BA.4.6 by mAbs used clinically

[0410] Finally, the neutralizing activity of a number of clinically useful mAbs was evaluated ( Fig.28 f). The potency of AZ1061 / cilgavimab against BA.4 / 5 is completely knocked out against BA.4.6, leading to a complete loss of activity of AZ7742 / Evusheld (a combination of AZ1061 / cilgavimab and AZ8895 / tixagevimab, which are already inactive against BA.4 / 5). The activity of S309 / sotovimab (which is no longer authorized by the US Food and Drug Administration for COVID-19 treatment as of April 2022 due to its ineffectiveness against BA.2) is further reduced compared to BA.2 and BA.4 / 5. Therefore, this leaves Ly-Cov1404 / betelovir as the only option for treating BA.4.6.

[0411] In summary, BA.4.6 showed further reduction in neutralization compared to BA.4 / 5 in sera from three-dose Pfizer vaccine recipients and BA.1 and BA.2 vaccine breakthrough recoveries. Notably, BA.4.6 did not appear to be more resistant to neutralization by sera from BA.4 / 5 breakthrough infections compared to the other variants. Altogether this suggests that unless you have been triple vaccinated and recovered from a BA.4 / 5 infection, you are very likely to be infected or have a breakthrough infection with BA.4.6, which appears to offer some protection against BA.4.6.

[0412] As of September 2022, bivalent booster vaccinations combining the ancestral strain with Omicron BA.1 are being rolled out in the UK and have recently been authorized by the FDA. The effectiveness of these bivalent boosters in preventing BA.4.6 infection remains to be seen. Finally, BA.4.6 further attenuates the activity of EvuSheld, which remains active against BA.4 / 5; therefore, only LY-CoV1404 / betecomab now remains effective against all circulating SARS-CoV-2 variants.

[0413] Systematic themes of mAb interactions

[0414] Omi-3 (a representative of the IGVH3-53 gene family) and AZD8895 (IGVH1-58) are both associated with F486. Although the F486V mutation has little effect on Omi-3 ( Fig.10 F, G, 11F), but it severely reduced the neutralization of AZD8895 and other IGVH1-58 mAbs (e.g., Omi-12) ( Fig.10 D, E, 11E). It is noteworthy that although many Omi series antibodies (9 out of 28, Figure 8 A, Table 21) are almost completely adapted to changes in BA.4 / 5, but the majority of antibodies generated against early variants from these gene families are knocked out on BA.1 and BA.2 (Nutalai et al., 2022), consistent with selection of a subset of antibodies insensitive to further BA.4 / 5 mutations by breakthrough Omicron infection.

[0415] The effects on antibodies with broadly similar epitopes can be quite different, and this is equally true for antibodies with either 452 or 486 centered in their binding footprints. Thus, Omi-31 (IGVH1-69) and Omi-32 (IGVH3-33) both bind in the anterior portion of the right shoulder, with their CDR-H3 positioned close to 452, while the activity of Omi-31 is abolished by L452R (as described above) and significantly enhanced by Omi-32 ( Figure 8 A, 11A, B). Similarly, Omi-25 and Omi-42 both belong to the IGVH3-9 gene family, and their footprints are both in the 486 region ( Fig.11 C, D). Omi-25 contacts F486, and thus neutralization of BA.4 / 5 is abolished. In contrast, Omi-42 does not contact either mutant site, and neutralization of BA.4 / 5 is fully preserved ( Fig.10 H, I, 11D).

[0416] Refined mapping of RBD antibody binding using competition measurements.

[0417] A matrix of pairwise BLI measurements was used to map the potent RBD binding of the Omicron mAb and several pre-pandemic mAbs with known binding locations.

[0418] The approach produced consistent predictions. The mAbs separated into a limited set of epitopes that appear to be a subset of those observed against early pandemic viruses and are distinct from those observed against Beta. Essentially, the antibodies clustered in two regions, one that included VH3-53 and VH3-66 type antibodies toward the back of the neck / left shoulder, extending to the top of the left shoulder, and the other in the anterior portion of the right shoulder region of the neck, spreading toward the S309 known antibody binding site. This region was occupied by antibodies from the VH1-69 family, with the exception of Omi-2, which was located within another cluster. mAb Omi-09 (which showed reduced neutralization of Beta and Gamma) was located near residue 484, which was mutated from Glu to Lys in Beta / Gamma and to A1a in Omicron. VH1-69 mAbs Omi-24, 30, 31, and 34 (which showed reduced neutralization of Delta) were located near residue 452, which was mutated from Leu to Arg in Delta.

[0419] Structure of the anti-Omicron Fab / RBD complex

[0420] Structural analysis of selected potent Omicron mAbs. The crystal structures of Omicron BA.1 RBD in complex with three different Fabs were determined: Omi-3, 9, and 12. The resolution of the complex with Omi-12 was very low. Therefore the structure of the individual Fabs was determined at high resolution and subjected to rigid body fitting to obtain the complex structure.

[0421] Omi-3 belongs to the VH3-53 gene family and demonstrates how this gene family is adapted to broadly neutralize all major SARS-CoV-2 variants (however, like all potent Omicron antibodies, it does not bind to the SARS-CoV-1 RBD). A fundamental problem with these antibodies is that most VoCs have an N501Y mutation that introduces a steric clash with the LC CDR1 (L1), abrogating the binding of most VH3-53-containing antibodies. However, two mechanisms for replacing L1 to avoid this clash have been previously reported (Dejnirattisai et al., 2021b; Liu et al., 2021b). In mAb-222, isolated from an individual infected with an early pandemic strain, a proline was inserted at residue 30, which fills in Tyr-501 without clash (Dejnirattisai et al., 2021b), allowing it to effectively neutralize Alpha, Beta, and Gamma variants. Beta-27 uses an alternative mechanism that extends the HC CDR3 (H3) loop from the usual 9 to 11 residues, displacing L1 to create sufficient space to allow 501Y to be stabilized by main-chain interactions, thereby conferring similar cross-reactivity (Liu et al., 2021b).

[0422] Omi-3 uses the same mechanism as Beta-27 to accommodate the N501Y mutation, although the Omi-3 H3 is one residue longer. The other VH3-53 Omicron antibodies (Omi-18 and Omi-29) have H3s very similar to Beta-27 and may use the same mechanism. This L1 configuration is also compatible with the Y505H mutation in Omicron. However, neither 222 nor Beta-27 effectively neutralize Omicron, which may be due to specific features of the H3 loop that are tightly linked to the Q493R Omicron mutation.

[0423] Omi-9 is one of three VH3-30 mAbs and binds to the left shoulder of the RBD. Omi-9 showed relatively weak neutralization of Beta and Gamma (Figure 2). Other antibodies with high sequence similarity bind similarly, with H3 contacting residue 484. Although the resolution of the Omi-9 / BA.1 complex is lower However, it is clear that H3 contacts residue 484, explaining the sensitivity to E484K in Beta and Gamma, whereas E484A in Omicron is tolerated.

[0424] 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 glycosylation site at residue 102 of the heavy chain CDR3, the role of which is unclear. VH1-58 antibodies generated during early pandemic or betavirus infection showed reduced ability to neutralize Omicron, for example mAb 253, Beta-47, and known antibody D (AZD8895) showed reduced activity against Omicron BA.1 relative to Victoria, respectively.

[0425] In contrast, Omi-12 has adapted and can effectively neutralize Omicron and all VoCs ( Figure 2A , B). The VH1-58 antibody binds to the left shoulder epitope, H3 contacts S477N, but the mutation at this position in Iota has no effect on the neutralization of VH1-58mAb using pseudovirus assays. In addition, despite the T478K mutation, mAb 253 is still able to neutralize Delta. BA.2 and early pandemic mAb 150 (VH3-53). Detectable residual activity was observed in BA.1, BA.1.1 and BA.2 (BA.3 was not tested). Two complex structures were obtained in different space groups, which are very similar and provide 3 independent views of the complex. mAb 150 binds in a posture similar to the previously observed early pandemic virus, but it is translated and forms a looser interaction, which is consistent with the almost complete loss of neutralizing activity. This shows the huge impact of the adaptive mutations found in Omi-3.

[0426] Interestingly, in BA.2, the three serine residues that are mutated in the BA.1 RBD, namely S371L, S373P and S375F in the loop adjacent to the lipid binding pocket, are also mutated in BA.2, but the mutation at 371 is Phe, meaning that this may be a single point mutation of the early pandemic, while the S317L mutation in BA.1 requires two mutations. Therefore, BA.2 may have features in common with early versions of the Omicron lineage. In addition, the various views of this part of the structure provided show that it adopts a range of different conformations. This may be due to different crystal contacts and reflects the flexibility of this loop region. This may have biological function because the Ser mutation requires a double codon change and may affect the presentation of the RBD. Since we have multiple views of this loop in the early pandemic virus, VoC, Omicron BA.1 and BA.2, we can see that flexibility is maintained in all variants.

[0427] Modeling the impact of changes in BA.1, BA.1.1, and BA.2 on selected commercial known antibodies, early pandemic, and Beta mAbs

[0428] Known Antibodies A (REGN 10987) and B (10933): Known Antibody B (REGN 10933) binds to the posterior portion of the left shoulder, and REGN 10987 binds to the right shoulder. Both have activity knocked out by the Omicron lineage, with the exception of Known Antibody A (REGN 10987) with BA.2. Known Antibody B (REGN 10933) H2 contacts residue 493, and neutralizing activity against Omicron is universally lost due to the presence of Q493R in all Omicron strains. Known Antibody A (REGN 10987) H2 contacts residue 446. BA.2 uniquely lacks the G446S mutation, so regn10987 retains some neutralizing ability.

[0429] Known antibodies C (AZD1061) and D (AZD8895): Known antibodies C (AZD1061) and D (AZD8895) bind to the posterior of the left shoulder and the anterior of the right shoulder, respectively, and both show reduced neutralization. Known antibody C (AZD1061) is still able to neutralize BA.2 and BA.3 (reduced by about 10-fold), but neutralization of BA.1 is reduced by >100-fold compared to Victoria, and neutralization of BA.1.1 is reduced by >1000-fold compared to Victoria. Known antibody C (AZD1 061) is affected by contact with the G446S (not present in BA.2 and BA.3) and R346K (BA.1.1) mutations (contacted by L2 and H3). 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 that are prevalent in the Omicron lineage. Antibody E (AZD7442) (a combination of C and D) is known to retain some neutralizing activity against the Omicron strain as the sum of its components.

[0430] Known Antibodies F, G and H: All known antibodies F, G and H suffered a considerable loss of activity against Omicron. Known Antibodies F and H had a complete loss of activity, while known antibody G (ADG20) had a 276-fold reduction in activity against Omicron.

[0431] Known antibodies I and J: Both antibodies had knockout activity against the entire Omicron repertoire. Known antibody J (Ly-CoV1 6) (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 susceptible to the E484K mutation in delta but may tolerate E484A, but it also contacts residue 493, so the ubiquitous Omicron Q493R mutation will globally abolish binding.

[0432] Known Antibody K (S309): Known Antibody K (S309) retains reasonable activity across the Omicron lineage. S309 contacts H3 on the right flank that contacts G339 and N343 glycans, which are close to mutations in Serines 371, 373, and 375. The S371F mutation in BA.2 (but not S371L) may affect binding, resulting in slightly reduced activity against this virus.

[0433] Structure and ACE2 affinity of BA.2RBD

[0434] The affinity of Omicron BA.1, BA.1.1, BA.2 and BA.3 RBD to ACE2 was measured by SPR and BLI. The affinity of BA.1 is comparable to that of the early virus, 8nM and 7nM respectively (the binding affinity of Omicron RBD is shown in Tables 14 and 18), which means that the increased affinity conferred by S477N, Q498R and N501Y is offset by other mutations in the ACE2 footprint. Compared with the early virus, the affinity of BA.2 is slightly increased (about 1.5 times x and Y nM, respectively). Based on early measurements of the contribution of single mutations to binding affinity, G496S and triple mutations S371L, S373P and S375F reduce binding by 2 times and 2.2 times, respectively, while BA.2 lacks G496S and has S371F. This may be the reason for some of the differences, but it is more likely that mutations on the edge of the ACE2 footprint in BA.2 may enhance binding. This was confirmed by the structure of BA.2 / ACE2.

[0435] BA.4 / 5 RBD and ACE2 affinity

[0436] The affinity of BA.4 / 5RBD for ACE2 was also measured by SPR ( Fig.12AD). The affinity of BA.4 / 5RBD is increased compared to the ancestral virus, BA.1 and BA.2 (approximately 3-fold, 3-fold and 2-fold, respectively (BA.4 / 5 / ACE2 KD = 2.4 nM)) (Deinirattisai et al., 2022; Nutalai et al., 2022), which is mainly due to an increased binding half-life. Modeling of the ACE2 / RBD complex suggests that most of this effect comes from electrostatic complementarity between ACE2 and the RBD caused by the L452R mutation ( Fig.12 EG).

[0437] BA.2.45RBD and ACE2 affinity

[0438] 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 subnanomolar affinity (BA.2.75 / ACE2 KD = 0.45 nM) ( Fig.18 A, B). This represents a significant increase in affinity (9-fold) compared to BA.2 ( Fig.18 C), even tighter (5 times) than BA.4 / 5 ( Fig.18 D), which binds to ACE2 with higher affinity than BA.2 (Tuekprakhon et al., 2022). BA.2.75 was found to be the strongest ACE2 binder among all SARS-CoV-2 VoCs, including Alpha (Alpha / ACE2 KD = 1.5 nM; Fig.18 E), and is the first to bind SARS-CoV-2 VoC with subnanomolar affinity.

[0439] BA.2+N460K RBD could not be expressed, but the binding affinity of BA.2+R493Q RBD to ACE2 was also measured ( Fig.18 F) (KD = 0.55 nM). This confirms that the R493Q back mutation contributes to the high affinity of BA.2.75RBD.

[0440] Effects of mutations in BA.2.75

[0441] Compared to BA.2, the cluster of mutations in BA.2.75 has opposite effects on neutralization. The reversion mutation R493Q makes the virus more susceptible to neutralization using vaccine sera (the vaccine contains Q493), while N460K reduces neutralization titers to a greater extent when expressed alone than the combination of mutations observed in BA.2.75. N460K is a novel substitution that has not appeared in previous SARS-CoV-2 variants. This mutation was introduced into the BA.2 backbone and its effect on neutralization by BNT162b2 sera was evaluated. Strikingly, BA.2+N460K showed a 3.1-fold reduction in titers compared to BA.2, which is greater than the reduction observed in BA.2.75 and comparable to the reduction observed in BA.4 / 5.

[0442] Using a panel of potent mAbs derived from vaccinated individuals who suffered breakthrough infection with the BA.1 vaccine, we showed that many mAbs belonging to the IGHV3-53 / 66 family had reduced or knocked-out activity against BA.2.75. IGHV3-53 / 66 is the most commonly isolated mAb in SARS-CoV-2 and binds to an epitope on the “neck.” Therefore, it is not surprising that IGHV53 / 66 forms the major public antibody response and that the virus has evolved to evade this response.

[0443] Although the BA.2+N460K RBD could not be expressed, a previous study using yeast display showed that N460K could enhance the binding of RBD to ACE2, an effect similar to the N501Y mutation first described in Alpha (Zahradnik et al., 2021). Therefore, N460K could enhance antibody escape and also increase receptor binding affinity.

[0444] Interestingly, BA.2.75 also acquired the R493Q reversion mutation (Q493R was acquired in BA.1 and is present in all other Omicron sublineages except BA.4 / 5). The BA.2.75RBD is able to bind ACE2 with 9-fold higher affinity than BA.2 and more tightly than BA.4 / 5 (Dejnirattisai et al., 2022; Tuekprakhon et al., 2022). This is in part due to the R493Q mutation. The BA.2.75RBD has the highest receptor binding affinity of all SARS-CoV-2 variants measured to date.

[0445] These data suggest that there may be a delicate balance between antibody escape and ACE2 receptor affinity. Mutations in BA.2.75 result in reduced neutralizing titers of vaccine sera compared to BA.2. Single BA.2.75 mutations can result in greater reductions in neutralizing titers compared to the full BA.2.75S sequence, but these mutations are balanced by the R393Q back mutation, which has been selected to increase affinity for ACE2 and increase the transmissibility of BA.2.75.

[0446] BA.2.11, BA.2.12 and BA.2.13 RBD and ACE2 affinity

[0447] To evaluate possible changes in the transmissibility of BA.2 subvariants, SPR experiments were performed to analyze their binding to the RBD of ACE2 ( Fig. 27 dg). The affinity of these three RBD variants for ACE2 is approximately 3 nM, slightly higher than 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 be due to a slight improvement in complementarity between ACE2 and RBD due to mutations at leucine 452. Therefore, these variants may have a subtle advantage over BA.2 in terms of transmission.

[0448] Antigenic mapping of BA.3 and BA.4 / 5

[0449] The neutralization data described above have been used to place BA.3 and BA.4 / 5 on the antigenic map. The approach used to analyze the Delta and Omicron variants (Liu et al., 2021, "Reduced neutralization of SARS-CoV-2B.1.617 by vaccine and convalescent serum." Cell 184, 4220-4236e4213) was repeated, where individual viruses were modeled independently, allowing serum-specific scaling of responses. The measured and modeled responses were Fig.13 A (with 1551 observations and 340 parameters, and a residual error of 23%). The results are best visualized in three dimensions (see Fig.13 2D projection of B). This shows that, as expected, the Omicron sublineages cluster together but are well separated from the early pandemic viruses and the earlier VoC. Within the Omicron cluster, BA.4 / 5 are the most distant from the pre-Omicron viruses.

[0450] Antigenic mapping of BA.2.75

[0451] Neutralization of BA.2.75 was tested using sera from individuals previously infected during the pandemic. These included sera obtained early in the pandemic (before Alpha emerged), as well as sera obtained after infection with Alpha, Beta, Gamma, Delta, BA.1, and BA.2 ( Fig.25 ). As expected, BA.2.75 neutralization titers were lower than those of the homologous infecting strain (e.g., Alpha sera against Alpha virus). However, most striking was the complete loss of BA.2.75 neutralization using Delta sera (zero samples achieved 50% neutralization at a 1 / 20 dilution). However, in cases that had been vaccinated before or after Delta infection, titers to BA.2.75 were much higher.

[0452] These data were used to place BA.2.75 on a three-dimensional antigenic map using methods previously reported in Tuekprakhon et al., 2022 ( Fig. 22 A, B). Initially includes all VoC ( Fig. 22 A); this shows that BA.2.75 is grouped with the other Omicron viruses, which are separated into one hemisphere of the 3D plot. BA.2.75 appears to be completely separate from the other Omicron sublineages, and in particular from BA.4 / 5. It is also noteworthy that BA.2.75 and Delta are exactly opposite each other in the plot, emphasizing the antigenic distance between these two viruses. Because the data are high-dimensional and 3D projections may distort the true distances, they were calculated only for Omicron and early pandemic viruses (but full serological information was retained for each virus). The results are in Fig. 22 The main features of the full graph are shown and summarized in B, but allow the Omicron sublineages to be more widely distributed in 3D space. Notably, if the clustered early pandemic and BA.2 / BA.3 pairs are merged, the points are distributed as a triangular bipyramid, maximizing their separation, consistent with antigenic escape being an important factor in their evolution.

[0453] Example 3. Examples of antibodies that can be generated by swapping light chains between antibodies derived from the same heavy chain V gene

[0454] As discussed in the detailed description above, antibodies derived from the same heavy chain V gene can swap light chains to produce antibodies comprising the heavy chain variable region of a first antibody and the light chain variable region of a second antibody, and such new antibodies may have improved neutralization and / or other properties when compared to the "parent" antibody.

[0455] Tables 4 to 12 provide examples of such antibodies that can be generated by swapping 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 were derived, as well as their specificity for the RBD or NTD of the spike protein of SARS-CoV-2.

[0456] Example 4. Materials and methods

[0457] Virus stock solution

[0458] SARS-CoV-2 / human / AUS / VIC01 / 2020 (Caly et al., 2020) Alpha and Beta were provided by Public Health England, Gamma was cultured from a throat swab from Brazil, Delta was a gift from Wendy Barclay and Thushan de Silva from the UK G2P Genotype to Phenotype Consortium, and Omicron was cultured from a positive throat swab (IRAS Project ID: 269573, Ethics Reference: 19 / NW / 0730). Briefly, VeroE6 / TMPRSS2 cells (NIBSC) were maintained in Dulbecco's modified Eagle's medium (DMEM) high glucose supplemented with 1% fetal bovine serum, 2mM Glutamax, 100IU / ml penicillin-streptomycin and 2.5ug / ml amphotericin B at 37°C in the presence of 5% CO2, and then 200ul of swab liquid was inoculated. The cells were further maintained at 37°C and observed daily for cytopathic effect (CPE). The virus-containing supernatant was clarified at 80% CPE by centrifugation at 3,000 rpm at 4°C and then stored at -80°C in single-use aliquots. Virus titers were determined by focus formation assay on Vero CCL-81 cells (ATCC).

[0459] Sequencing of the Omicron isolate revealed the 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), an intact furin cleavage site, and a single additional mutation, A701V.

[0460] Cells were infected with SARS-CoV-2 virus using an MOI of 0.0001.

[0461] Supernatants containing virus were harvested at 80% CPE and spun at 3000 rpm at 4°C and then stored at -80°C. Virus titers were determined by focus formation assay on Vero cells. Victoria channel 5, Alpha channel 2, and Beta channel 4 stocks Gamma channel 1, Delta channel 3, and Omicron channel 1 were sequenced to verify that they contained the expected spike protein sequence and that the furin cleavage site was unchanged.

[0462] Bacterial strains and cell culture

[0463] Vero (ATCC CCL-81) and VeroE6 / TMPRSS2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2mM GlutaMAX (Gibco, 35050061) and 100U / ml penicillin-streptomycin at 37° C. Human mAbs were expressed in HEK293T cells cultured in UltraDOMA PF protein-free medium (Cat. No. 12-727F, LONZA) at 37° C. and 5% CO2. HEK293T (ATCC CRL-11268) cells were cultured in DMEM high glucose (Sigma Aldrich) supplemented with 10% FBS, 1% 100X Mem Neaa (Gibco) and 1% 100X L-glutamine (Gibco) at 37°C and 5% CO2. In order to express RBD, RBD variants and ACE2, HEK293T cells were cultured in DMEM high glucose (Sigma) supplemented with 2% FBS, 1% 100X Mem Neaa and 1% 100X L-glutamine at 37°C for transfection. Omicron RBD and human mAbs were also expressed in HEK293T (ATCC CRL-11268) cells cultured in FreeStyle 293 expression medium (ThermoFisher, 12338018) at 37°C and 5% CO2. E. coli DH5α bacteria were used for transformation and large-scale preparation of plasmids. Single colonies were picked and cultured in LB broth at 37°C in a shaker at 200 rpm overnight.

[0464] Serum from Pfizer vaccine recipients

[0465] Pfizer vaccine sera were obtained from volunteers who had received one or two doses of the BNT162b2 vaccine. Vaccine recipients were healthcare workers at Oxford University Hospitals NHS Foundation Trust who had not been previously infected with SARS-CoV-2 and were participating in the OPTIC study as part of the Oxford Translational Gastroenterology GI Biobank Study 16 / YH / 0247 [Yorkshire & The Humber-Sheffield Research Ethics Committee (REC)], which was amended for this purpose on 8 June 2020. The study 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. Written informed consent was obtained from all participants participating in the study. Participants were studied after receiving two doses of the Pfizer / BioNTech BNT162b2 mRNA vaccine (30 micrograms diluted intramuscularly (0.3 mL each)), and samples were collected approximately 28 days (range 25-38 days), 180 days (range 178-221 days), and 270 days (range 243-273 days) after receiving two doses of the vaccine, and then sampled 17-28 days and then approximately 28 days (range 25-56 days) after receiving the third booster dose of BNT162B2 vaccine. The average age of the vaccine recipients was 37 years (range 22-66 years), 21 men and 35 women.

[0466] Plasma from early pandemic and Alpha cases

[0467] Participants from the first wave of SARS-CoV2 in the UK and sequence-confirmed B.1.1.7 lineages in December 2020 and February 2021 were recruited through three studies: Sepsis Immunomics [Oxford REC C, Ref: 19 / SC / 0296], the ISARIC / WHO Clinical Characterization of Severe Emerging Infections protocol [Oxford REC C, Ref: 13 / SC / 0149], and the Oxford Gastrointestinal Diseases: COVID Sub-study [Sheffield REC, Ref: 16 / YH / 0247]. Diagnosis was confirmed by reporting symptoms consistent with COVID-19 and a positive upper respiratory tract (nose / throat) swab for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an accredited laboratory. Blood samples were collected with consent at least 14 days after symptom onset. Clinical information was captured for all individuals at the time of sampling, including severity of illness (classified as mild, severe, or critical infection, as recommended by the World Health Organization) and time between symptom onset and sampling, as well as the age of the participant. After heat inactivation of plasma / serum samples, they were aliquoted to undergo no more than three freeze-thaw cycles for data generation.

[0468] Sera from cases infected with Beta, Gamma, and Delta, and BA.1

[0469] Beta and Delta samples from UK infected cases were collected according to the protocol ‘Innate and adaptive immunity to SARS-CoV-2 in families and household members of healthcare workers’ affiliated with the Oxford Gastroenterology: COVID sub-study’ discussed above and approved by the Central University Research Ethics Committee of the University of Oxford. All individuals had sequence-confirmed Beta / Delta infection or PCR-confirmed symptomatic disease, which occurred during quarantine and when in direct contact with Beta / Delta sequence-confirmed cases. Additional Beta-infected sera (sequence-confirmed) were obtained from South Africa. At the time of swab collection, patients signed an informed consent form for data collection and serial blood samples. The study was approved by the Human Research Ethics Committee of the University of the Witwatersrand (reference number 200313) and was conducted in accordance with Good Clinical Practice guidelines. Gamma samples were provided by the Coronavirus International Reference Laboratory at FIOCRUZ (WHO) as part of national coronavirus surveillance and approved by the FIOCRUZ Ethics Committee (CEP 4.128.241) to continuously receive and analyse samples from suspected COVID-19 cases for virological surveillance. Clinical samples were shared with the University of Oxford, UK, under MTA IOC FIOCRUZ 21-02.

[0470] Sera from BA.1 infected cases, study subjects

[0471] After informed consent, individuals with omicron BA.1 were co-enrolled in the ISARIC / WHO Clinical Characterization of Severe Emerging Infections protocol [Oxford REC C, Ref: 13 / SC / 0149] and the 'Innate and Adaptive Immunity to SARS-CoV-2 in Families of Healthcare Workers and Household Members' protocol affiliated with the Oxford Gastrointestinal Disease: COVID Sub-study [Sheffield REC, Ref: 16 / YH / 0247] further approved by the Central University Research Ethics Committee of the University of Oxford. Diagnosis was confirmed by reporting symptoms consistent with COVID-19 or positive contact of a known omicron case and positive SARS-CoV-2 from an upper respiratory tract (nose / throat) swab tested by an accredited laboratory using reverse transcriptase polymerase chain reaction (RT-PCR) and lineage sequence confirmation by a national reference laboratory. Blood samples were collected with consent at least 10 days after PCR test confirmation. Clinical information was captured for all individuals at the time of sampling, including severity of illness (classified as mild, severe or critical infection, as recommended by the World Health Organization) and time between symptom onset and sampling, as well as the age of the participant.

[0472] AstraZeneca-Oxford vaccine research procedures and sample handling

[0473] Full details of the randomized controlled trial of ChAdOx1 nCoV-19 (AZD1222) have been published previously (PMID: 33220855 / PMID: 32702298). These studies are registered with ISRCTN (15281137 and 89951424) and ClinicalTrials.gov (NCT04324606 and NCT04400838). A copy of the protocol is included in the previous publication (Folegatti et al., 2020, Lancet 396, 467-478).

[0474] Data from vaccinated volunteers who received two vaccinations are included in the Examples. The vaccine dose was 5 × 10 10 The vaccine was administered as a single dose of 1000 viral particles (standard dose; SD / SD cohort n=21) or as a half dose as its first dose (low dose) and as a standard dose as its second dose (LD / SD cohort n=4). The interval between the first and second doses was in the range of 8-14 weeks. Blood samples were collected and serum was separated on the day of vaccination and on pre-specified days after vaccination (e.g., 14 and 28 days after booster).

[0475] Focus Reduction Neutralization Assay (FRNT)

[0476] The neutralization potential of Ab was measured using a focus reduction neutralization assay (FRNT), in which the reduction in the number of infected foci was compared with a negative control well without antibody. In brief, serially diluted Ab or plasma was mixed with SARS-CoV-2 strains and incubated at 37 ° C for 1 hour. The mixture was then transferred in duplicate to a 96-well cell culture-treated flat-bottom microplate containing a confluent Vero cell monolayer and incubated for another 2 hours, after which 1.5% semisolid carboxymethylcellulose (CMC) was added to each well to cover the medium to limit viral spread. The focus formation assay was then performed by staining Vero cells with human anti-NP mAb (mAb206) and peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, approximately 100 foci (infected cells) per well in the absence of antibody were visualized by adding TrueBlue peroxidase substrate. Virus-infected cell foci were counted on a classic AID EliSpot reader using AID ELISpot software. The percentage of lesion reduction was calculated and the IC50 was determined using the probit procedure from the SPSS software package.

[0477] Plasmid construction and pseudotyped lentiviral particle production

[0478] Pseudotyped lentiviruses expressing SARS-CoV-2 S protein from ancestral strains (Victoria, S247R), BA.1, BA.1.1, and BA.2 were constructed as previously described (Nie, Jianhui et al. "Establishment and validation of apseudovirus 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 some modifications. Briefly, synthetic codon-optimized SARS-CoV-2 BA.1 and BA.2 were custom synthesized by GeneArt (Thermo Fisher Scientific GENEART). Inserts and pcDNA3.1 vectors were cloned using Gibson assembly. Victoria (S247R) constructs were previously described in Liu, Chang et al. "Reduced neutralization of SARS-CoV-2B.1.617 by vaccine and convalescent serum." Cell 184.16 (2021): 4220-4236.To construct BA.1.1, PCR amplification was performed using the BA.1 construct as a template together with two primers of the pcDNA3.1 vector (pcDNA3.1BamHI_F 5′-GGATCCATGTTCCTGCTGACCACCAAG AG-3′ and pcDNA3.1_Tag_S_EcoRI_R 5′-GAATTCTCACTTCTCGAACTGAGGGTGGC-3′) to the mutagenic primers of R346K (R346K_F 5′-GTGTTCAATGCCACCAAATTCGCCAGCGTGTAC-3′ and R346K_R 5′-GTACACGCTGGCGAATTTGGTGGCATTGAACAC-3′), purified using a QIAquick gel extraction kit (QIAGEN), and then Gibson assembly was performed. After plasmids were isolated using a QIAGEN Miniprep kit (QIAGEN), all constructs were verified by Sanger sequencing.

[0479] A similar strategy was applied for BA.3 and BA.4 / 5. In brief, BA.3 mutations were constructed using combined fragments from BA.1 and BA.2. The resulting mutations are as follows. 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. Although the BA.4 / 5S protein shares some amino acid mutations with BA.2 (Nutalai et al., 2022), to generate the BA.4 / 5 mutations, Δ69-70, i.e., L452R, F486V, and R498Q, were added. The resulting pcDNA3.1 carrying the S gene was used to generate pseudoviral particles together with a lentiviral packaging vector and a transfer vector encoding a luciferase reporter. The integrity of the construct was sequence confirmed.

[0480] The same method is also used to construct BA.2.12.1, and BA.2.75 is constructed by adding more mutations to BA.2. In order to produce BA.2.75, K147E, W152R, F157L, I210V, G275S, G446S and N460K are added to the BA.2 backbone. 339D is also changed to 339H in BA.2 S, and 493R is backmutated to 493Q in BA.2, the same as the ancestral strain. In order to test the impact of single mutations, D339H, G446S, N460K and R493Q are introduced into the BA.2 backbone alone. The resulting pcDNA3.1 plasmid carrying the S gene is used to produce pseudoviral particles together with a lentiviral packaging vector and a transfer vector encoding a luciferase reporter.

[0481] Pseudovirus neutralization test

[0482] Details of the pseudovirus neutralization assay were described previously (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. In brief, potent monoclonal antibodies (mAbs) generated from donors who had recovered from Omicron and Beta infections and from donors infected during the early pandemic in the UK were assayed for neutralizing activity 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 pseudoviral particles at 37°C, 5% CO2 for 1 hour. Stable HEK293T / 17 cells expressing human ACE2 were then added to the mixture at 1.5 x 104 cells / well. 48 hours after transduction, the culture supernatant was removed and 50 μL of 1:2 Bright-GloTM luciferase assay system (Promega, USA) in 1x PBS was added to each well. The reaction was incubated at room temperature for 5 minutes and the cells were incubated using Firefly luciferase activity was measured (BMG Labtech, Ortenberg, Germany). mAb neutralization percentage was calculated relative to control. Probit analysis was used to estimate the dilution that inhibits half-maximal pseudotyped lentiviral infection (PVNT50).

[0483] To determine the neutralizing activity of convalescent plasma / serum samples or vaccine sera, 3-fold serial dilutions of the samples were incubated with pseudovirions for 1 h and the same strategy as for mAbs was applied.

[0484] DNA manipulation

[0485] Cloning was performed by using the unrestricted method (Peleg and Unger, 2014). PCR amplification was performed with mutagenic megaprimers (KAPA HiFi HotStart ReadyMix, Roche, Switzerland, catalog number KK3605) using The protein was purified by gel and PCR purification kit (Nacherey-Nagel, Germany, REF 740609.50) and cloned into pJYDC1 (Adgene ID: 162458) (Zahradnik et al., 2021a). The parental pJYDC1 molecule was cut by DpnI treatment (1 h, New England Biotechnology (NEB, USA), catalog number R0176), and the reaction mixture was electroporated into Escherichia coli. 10G cells (Lucigen, USA). The correctness of the mutagenesis was verified by sequencing.

[0486] Cloning of the spike protein and RBD

[0487] Expression plasmids for wild-type and Omicron BA.1 spike proteins and the RBDs of BA.1 and BA.2 encoding the human codon-optimized sequences of BA.1 (EPI_ISL_6640917) and BA.2 (EPI_ISL_6795834.2) were constructed. The construction of wild-type and BA.1 spike proteins and RBD plasmids was the same as previously described (Dejnirattisai, Wanwisa et al. "The antigenic anatomy of SARS-CoV-2 receptor binding domain." Cell 184.8 (2021): 2183-2200). A synthetic codon-optimized RBD fragment of BA.2 was used as a template, and the constructs were amplified by PCR and cloned into the pNEO vector as previously described (Dejnirattisai et al., 2021a; Supasa et al., 2021; Zhou et al., 2021). The construct was verified by Sanger sequencing.

[0488] To generate the His-tagged BA.4 / 5RBD construct, site-directed PCR mutagenesis was performed using the BA.2RBD construct as a template ( Nutalai et al., 2022 ), in which L452R, F486V, and R493Q mutations were introduced. The gene fragments were amplified using pNeoRBD333Qmi|F (5′-GGTTGCGTAGC TGAAACCGGTCATCACCATCACCATCACACCAATCTGTGCCCT TTCGAC-3′) and pNeoRBD333_R (5′-GTGATGGTGGTGCTTGGTACCT TATTACTTCT TGCCGCACACGGTAGC-3′) and cloned into the pNeo vector (Supasa et al., 2021, “Reduced neutralization of SARS-CoV-2B.1.1.7 variant by convalescent and vaccine sera”. Cell 184, 2201-2211e2207). To generate the BA.4 / 5RBD construct containing the BAP-His tag, gene fragments were amplified with RBD333F (5′-GCGTAGCTGAAACCGGCACCAATCT GTGCCCTTTCGAC-3′) and RBD333BAP R (5′-GTCATTCAGCAAGC TCTTCTTGCCGCACACGG TAGC-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). After plasmid isolation using QIAGEN Miniprep kit (Qiagen), the construct was verified by Sanger sequencing.

[0489] To generate the BA.2.75RBD construct, site-directed PCR mutagenesis was performed using the BA.2 spike protein construct as a template (Nutalai et al., 2022) using Fig.26The primers listed in introduced D339H, G446S, N460K and R493Q mutations; D339H_pNeoF and RBD333_BAP_R ( Fig.26 ) was used to amplify the gene fragment 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, site-directed PCR mutagenesis was performed using the BA.2 spike protein construct as a template, using Fig.26 The R493Q mutation was introduced using the primers listed in ; the gene fragment was 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, 2201-2211e2207). Cloning was performed using the ClonExpress II one-step cloning kit (Vazyme). After isolating the plasmid using the QIAGEN Miniprep kit (Qiagen), the construct was verified by Sanger sequencing.

[0490] Production of RBD

[0491] The plasmid encoding RBD was transfected into Expi293FT by PEI. M Cell (Thermo Fisher Scientific), in FreeStyle TM 293 expression medium (Thermo Fisher) at 30 ° C and 8% CO 2 for 4 days. To express biotinylated RBD, the RBD-BAP plasmid was co-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).

[0492] BA.2.75 Generation of RBD

[0493] The plasmid encoding RBD was transfected into Expi293FT by PEI. M Cell (Thermo Fisher Scientific), in FreeStyle TM 293 expression medium (Thermo Fisher) at 37 ° C and 8% CO2 for 1 day, and then at 30 ° C for 3 days. To express biotinylated RBD, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20856; encoding ER-localized biotin ligase) in the presence of 0.8 mM D-biotin (Sigma Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBD was purified by His tag binding using a 5 mL HisTrap nickel column (GE Healthcare), followed by purification using a Superdex 7510 / 300GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.

[0494] Protein production

[0495] Protein expression and purification were performed as previously described (Dejnirattisai et al., 2021a; Zhou et al., 2020). In brief, plasmids encoding proteins were transiently expressed in HEK293T (ATCCCRL-11268) cells. Conditioned media was concentrated using a QuixStand benchtop system. His-tagged Omicron RBD was purified with a 5 mL HisTrap nickel column (GE Healthcare) and further purified using a Superdex 75HiLoad 16 / 60 gel filtration column (GE Healthcare). The double-streptococcal-tagged Omicron spike protein was purified with Strep-Tactin XT resin (IBA Lifesciences). About 4 mg of ACE2 was mixed with homemade His-tagged 3C protease and DTT (final concentration 1 mM). After incubation at 4 ° C for one day, the sample was passed through a 5 mL HisTrap nickel column (GE Healthcare). His-tagged proteins were removed by a nickel column, and purified ACE2 was harvested and concentrated.

[0496] IgG mAb and Fab purification

[0497] To purify full-length IgG mAb, the supernatant of mAb expression was collected and filtered through a vacuum filtration system and loaded onto protein A / G beads at 4°C overnight. The beads were washed three times with PBS and IgG was eluted using 0.1M 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 spectrophotometrically and buffer exchanged into PBS. To express and purify Fab 158 and EY6A, the heavy and light chain expression plasmids of Fab were co-transfected into HEK293T cells by PEI. After the cells were cultured at 37°C and 5% CO2 for 5 days, the culture supernatant was harvested and filtered using a 0.22mm polyethersulfone filter. Fab 158 was purified using Strep-Tactin XT resin (IBA Life Sciences), and Fab EY6A was purified using a Ni-NTA column (GE Healthcare) and a Superdex 75HiLoad 16 / 60 gel filtration column (GE Healthcare). AstraZeneca and Regeneron antibodies were provided by AstraZeneca, and Vir, Lilly, and Adagio antibodies were provided by Adagio. For these antibodies, the heavy and light chains of the indicated antibodies were transiently transfected into 293Y cells, and the antibodies were purified from the supernatant on protein A. Fab fragments of 58 and β-55 were digested with papain from purified IgG using the Pierce Fab preparation kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0498] Surface Plasmon Resonance

[0499] Surface plasmon resonance experiments were performed using Biacore T200 (GE Healthcare). All measurements were performed at 25°C using HBS-EP (Sterofam) running buffer.

[0500] To determine the binding kinetics between SARS-CoV-2 RBD and ACE2 / monoclonal antibody (mAb), a Protein A sensor chip (Science & Technology Corporation) was used. ACE2-Fc or mAb was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left empty. A single cycle kinetic program was used at 30 μl min -1 RBD was injected into two flow cells at a flow rate of 1.5 Å at a series of five concentrations prepared by serial two-fold dilutions. Running buffer was also injected using the same procedure to subtract background. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0501] To determine the binding kinetics between the SARS-CoV-2 spike protein and ACE2, a CM5 sensor chip was used. The sensor chip was first activated by injecting an equal volume of a mixture of EDC and NHS (Situofan) at 20uL / min over 300s, then a 20ug / mL spike protein sample (Situofan) in 10mM sodium acetate pH 5.0 was injected into the sample flow cell of the sensor chip at 10uL / min, and finally 1.0 M ethanolamine-HCl pH 8.5 (Situofan) was injected at 20uL / min over 180s. The reference flow cell was left empty. Using a single cycle kinetic program, the flow cell was quenched at 30μl min -1 ACE2 was injected into two flow cells at a flow rate of 1.5 Å at a series of five concentrations prepared by serial two-fold dilutions. Running buffer was also injected using the same procedure to subtract background.

[0502] All data were fitted to a 1:1 binding model using Biacore T200 evaluation software 3.1. To determine the binding kinetics between RBD and mAb Omi-32 / Omi-42, a biotin CAPture kit (Situofan) was used. The biotinylated RBD was fixed to the sample flow cell of the sensor chip. The reference flow cell was left empty. Using a single cycle kinetic program, mAb Fab was injected into two flow cells at a flow rate of 30 μl min-1 with a series of five concentrations prepared by serial two-fold dilution. The same program was also used to inject running buffer to subtract background. All data were fitted to a 1:1 binding model using Biacore T200 evaluation software 3.1.

[0503] To determine the binding affinity of BA.4 / 5RBD and mAb Omi-12, a protein A sensor chip (Situofan) was used. Ig Omi-12 was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left empty. At a flow rate of 30 μl min-1, RBD was injected into two flow cells in a series of seven concentrations prepared by serial two-fold dilution. The same procedure was also used to inject running buffer to subtract background. All data were fitted to a 1:1 binding model using Prism9 (GraphPad).

[0504] In order to compare the binding of BA.2 and BA.4 / 5RBD to mAb Omi-06 / Omi-25 / Omi-26, a protein A sensor chip (Situofan) was used. The mAbs in IgG form were fixed to the sample flow cell of the sensor chip to a similar level (about 350RU). The reference flow cell was left empty. A single RBD injection was performed at 200nM on both flow cells at a flow rate of 30μl min-1. The running buffer was also injected using the same procedure to subtract the background. The sensorgram was drawn using Prism9 (GraphPad).

[0505] To compare the binding of BA.2 and BA.4 / 5RBD to mAb Omi-02 / Omi-23 / Omi-31, a biotin CAPture kit (Situofan) was used. Biotinylated BA.2 and BA.4 / 5RBD were fixed to the sample flow cell of the sensor chip to similar levels (about 120RU). The reference flow cell was left empty. A single mAb Fab injection was performed on both flow cells at a flow rate of 30 μl min-1 at 200nM. The same procedure was also used to inject running buffer to subtract background. Sensorgrams were drawn using Prism9 (GraphPad).

[0506] To determine the binding kinetics between BA.2.75 or BA.2+R493Q RBD and ACE2, a protein A sensor chip (Situofan) was used. ACE2-Fc was fixed to the sample flow cell of the sensor chip. The reference flow cell was left empty. Using a single cycle kinetic program, RBD was injected into two flow cells at a flow rate of 30 μl min-1 with a series of five concentrations prepared by serial two-fold dilution. The same program was also used to inject running buffer to subtract background. All data were fitted to a 1: 1 binding model using Biacore T200 evaluation software 3.1.

[0507] To confirm the binding kinetics between BA.2.75RBD and ACE2, a biotin CAPture kit (Situofan) was used. Biotinylated ACE2 (bio-ACE2) was fixed to the sample flow cell of the sensor chip. The reference flow cell was left empty. A single cycle kinetic program was used at 30 μl min -1 BA.2.75RBD was injected into two flow cells at a flow rate of 1.5 Å in a series of five concentrations prepared by serial two-fold dilutions. Running buffer was also injected using the same procedure to subtract background. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0508] To determine the binding kinetics between BA.2.75 or BA.2 RBD and mAb, a biotin CAPture kit (Science & Technology) was used. The biotinylated RBD was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left empty. A single cycle kinetic program was used at 30 μl min -1 Fabs of Omi-18 or Omi-32 were injected into both flow cells at a series of five concentrations prepared by serial two-fold dilutions at a flow rate of 1.5 Å. For binding of Omi-20 to bio-BA.2 RBD, a single cycle kinetic program was used at 30 μl min -1 The Fab of Omi-20 was injected into two flow cells at a series of five concentrations prepared by serial two-fold dilutions at a flow rate of 30 μl min. -1 At a flow rate of 1.5 Å, Fab of Omi-20 was injected into two flow cells at a series of eight concentrations prepared by serial two-fold dilutions. Running buffer was also injected using the same procedure to subtract background. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

[0509] To compare the binding of BA.2 and BA.2.75RBD to mAb Omi-29, a biotin CAPture kit (Science & Technology Corporation) was used. Biotinylated BA.2 and BA.2.75RBD were immobilized to the sample flow cell of the sensor chip to similar levels (approximately 110 RU). The reference flow cell was left empty. -1 A single mAb Fab injection was performed at 1 μM on both flow cells at a flow rate of 1 μM. Running buffer was also injected using the same procedure to subtract background. Sensorgrams were plotted using Prism 9 (GraphPad).

[0510] To compare the binding of BA.2 and BA.2.75 RBD to mAb Omi-36, a sensor chip Protein A (Science) was used. mAb Omi-36 in IgG form was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left empty. -1 A single RBD injection was performed at 200 nM on both flow cells at a flow rate of 1.5 ng / mL. Running buffer was also injected using the same procedure to subtract background. Sensorgrams were drawn using Prism 9 (GraphPad).

[0511] IgG mAb and Fab production

[0512] AstraZeneca and Regeneron antibodies were provided by AstraZeneca, and Vir, Lilly, and Adagio antibodies were provided by Adagio, and LY-CoV1404 was provided by LifeArc. For in-house antibodies, the heavy and light chains of the indicated antibodies were transiently transfected into 293Y or 293T cells, and the antibodies were purified from the supernatant on protein A as previously described (Nutalai et al., 2022). Fabs were digested from purified IgG with papain using the Pierce Fab preparation kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0513] Quantification and statistical analysis

[0514] Statistical analysis is reported in the results and figure legends. Neutralization was measured by FRNT. The percentage of lesion reduction was calculated and IC50 (FRNT50) was determined using the probit program from the SPSS software package. Analysis was performed using the Wilcoxon paired signed rank test, and two-tailed P values ​​were calculated based on the geometric mean.

[0515] crystallization

[0516] The RBD protein was deglycosylated with endoglycosidase F1 before being used for crystallization. Omicron BA.1-RBD was mixed with Omi-12 and beta-54 Fab in a molar ratio of 1:1:1, respectively, with a final concentration of 7 mg ml-1. The complexes were incubated at room temperature for 30 minutes. A preliminary screen of crystals was set up in a Crystalquick 96-well X plate (Greiner Bio-One) using a Cartesian robot using nanoliter sitting drop vapor diffusion, with 100 nL protein plus 100 nL reservoir in each drop, as previously described (Walter et al., 2003, Journal of Applied Crystallography 36, 308-314).

[0517] Crystals of BA.1-RBD / Omi-12 / beta-54 were formed under Hampton Research PEGRx Condition 1-46 containing 0.1 M trisodium citrate dihydrate pH 5.0 and 18% (w / v) PEG 20000. The complex of BA.1-RBD / Omi-12 / beta-54 was screened under Hampton Research ammonium sulfate screen C2 containing 2.4 M (NH4)2SO4 and 0.1 M citric acid pH 5.0, but only crystals of Fab Omi-12 alone were formed under this condition.

[0518] BA.2.75 RBD crystal

[0519] The purified BA.2.75RBD was deglycosylated using endoglycosidase H1 and mixed with ACE2 at a 1:1 molar ratio to a final concentration of 13.0 mg ml -1 Initial screening of crystals was set up using nanoliter sitting drop vapor diffusion with a Cartesian robot in Crystalquick 96-well X plates (Greiner Bio-One) with 100 nL protein plus 100 nL reservoir in each drop as previously described (Walter et al., 2003). Crystals of the BA.2.75RBD-ACE2 complex were formed under Hampton Research PEGRx conditions 2-25 containing 0.1% (w / v) n-octyl-bD-glucoside, 0.1 M trisodium citrate dihydrate pH 5.5, and 22% (w / v) PEG 3350. Diffraction data were collected at 100 K at beamline I03 at Diamond Light Source (UK) using an automated queue system that allows unattended automated data collection (https: / / www.diamond.ac.uk / Instruments / Mx / I03 / I03-Manual / Unattended-Data-Collections.html).

[0520] X-ray data collection, structure determination and refinement

[0521] Diffraction data were collected at 100 K at beamline I03 at Diamond Light Source, UK. All data were collected as part of an automated queue system that allows unattended automated data collection (https: / / www.diamond.ac.uk / Instruments / Mx / I03 / I03-Manual / Unattended-Data-Collections.html). Crystals were pre-frozen by mounting them in a ring and immersing them for one second in a cryoprotectant containing 25% glycerol and 75% mother liquor. Diffraction images were recorded on an Eiger2 XE 16M detector with 0.1° rotation (exposure time 0.018 s per image, beam size 80 × 20 μm, beam transmission 10%, wavelength 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). 360° data were collected from a single crystal for each data set.

[0522] The structure was determined by molecular replacement using PHASER (McCoy et al., 2007, J Appl Crystallogr 40, 658-674). The structure of the SARS-CoV-2 RBD / Fab will be compared with the previously determined SARS-CoV-2 RBD / Fab structure (Dejnirattisai et al., 2021, Cell, 184, 2183-2200e2122; Dejnirattisai et al., 2021, Cell, 184, 2939-2954e2939; Huo et al., 2020, Cell Host Microbe, 28, 445-454; Liu et al., 2021, Cell, 184, 4220-4236e4213; Supasa et al., 2021, Cell, 184, 2201-2211e2207; Zhou et al., 2021, Cell, 184, 2348-2361e2346; Zhou et al., 2020, Nature structural & molecular Biology [Nature Structural and Molecular Biology] 27, 950-958) The VhVl and ChCl domains with the greatest sequence similarity were used as search models for each current structure determination.

[0523] All structures were rebuilt using COOT models (Emsley et al., 2010, BiologicalCrystallography 66, 486-501) and refined using Phenix (Liebschner et al., 2019, ActaCrystallogr D Struct Biol 75, 861-877). Due to the low resolution, only the structure of the BA.1-RBD / O-12 / Beta-54 complex was refined with rigid body and group B factors.

[0524] Data collection and structure refinement statistics 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 the PyMOL (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrodinger & Co., Ltd. LLC)) prepared the attached drawings.

[0525] Example 5

[0526] Antibody structure

[0527] The structure of the BA.1RBD / Fab Omi-12 / Fab Beta-54 ternary complex was determined to be Resolution (Table 19, Figure 6 A). Although BLI experiments showed no significant competition between the two Fabs for binding, slight clashes between them were observed. The high-resolution structure of the uncomplexed Omi-12fab has been resolution, Table 19) was modeled as the electron density of the complex ( Figure 6 B, 6C). Superposition of Fab 253 onto Fab Omi-12 showed that Q493R would clash with the H2 loop of Fab 253, whereas in Omi-12, H2 adopts a slightly flattened structure. This structural change can be attributed to the maturation of the antibody via the somatic mutation V53P in the heavy chain variable region of Omi-12, which forms a stacking interaction with Y489 ( Figure 6 D).

[0528] Omi-12 and antibody 253 are both derived from the germline heavy chain IGHV1-58. Interestingly, similar to antibody 253, the other antibodies described herein that are derived from the germline heavy chain IGHV1-58, namely Beta-47, Beta-25, Antibody 55, Antibody 165 and Antibody 318, also have a valine (V) at position 53 in the heavy chain variable region, namely 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 these data, modification of any of these antibodies by substituting proline for valine at position 53 (ie, V53P by absolute numbering, or V58P by IMGT numbering) would result in antibodies that are effective against Omicron.

[0529] In addition, antibody AZD8895 (heavy chain variable region amino acid sequence provided in SEQ ID NO: 963, and light chain variable region amino acid sequence provided in SEQ ID NO: 965) is also derived from germline heavy chain IGHV1-58 (e.g., Dong et al., Nat Microbiol [Nature Microbiology] 6, 1233-1244 (2021)). AZD8895 has an isoleucine (I) at position 53 (corresponding to position 58 according to IMGT numbering) in the heavy chain variable region. Based on the data herein, modifying the heavy chain variable region AZD8895 (SEQ ID NO: 963) by replacing the isoleucine at position 53 with proline (i.e., using absolute numbering of I53P, or using IMGT numbering of I58P) will produce an antibody that is effective against Omicron.

[0530] Thus, the data suggest that modifying the VH1-58 antibodies so that there is a proline at position 53 (corresponding to position 58 according to IMGT numbering) in the heavy chain variable region will render them particularly effective against Omicron.

[0531] ACE2 / BA.2.75 RBD structure

[0532] To elucidate the molecular mechanism of high affinity, the structure of BA.2.75RBD with ACE2 was determined by crystallography (according to the methods described in Example 4). As expected, the binding mode was essentially indistinguishable from that observed previously ( Fig. 20A), despite significant rearrangements outside the ACE2 footprint, where the flexible RBD 371-375 loop rearranges and a partial C-terminal 6xHis tag becomes ordered. Fig. 20 B shows a close-up of the binding interface compared to the ACE2 / BA.2RBD complex. In other complexes (with R or Q at RBD 493), K31 of ACE2 tends to be disordered, while in the BA.2.75 complex it is well-ordered, allowing K31 to form potential hydrogen bonds with the glutamine side chain, which may increase the affinity of ACE2.

[0533] surface

[0534] Table 1 - SEQ ID NOs of antibodies raised against early pandemic strains

[0535]

[0536]

[0537] Table 2 - SEQ ID NOs of antibodies raised against beta strains

[0538]

[0539]

[0540] Table 3 - SEQ ID NOs of antibodies raised against the Omicron strain

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549] Table 8 - Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV3-30

[0550]

[0551] Table 9 - Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV3-33

[0552]

[0553] Table 10 - Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV1-18

[0554]

[0555] Table 11 - Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV3-9

[0556]

[0557] Table 12 - Examples of mixed chain antibodies generated from antibodies derived from the same germline heavy chain IGHV4-31

[0558]

[0559] Table 13-22 Omicron SARS-CoV-2 specific human mAbs against live virus strains Victoria, Alpha, IC50 titers of Beta, Gamma, Delta and Omicron (BA.1).

[0560]

[0561]

[0562]

[0563] Table 14-22 Omicron SARS-CoV-2 specific human mAbs against pseudovirus strains Victoria, Omicron IC50 titers for BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3.

[0564]

[0565] Table 15 - Early pandemic SARS-CoV-2 specific human mAbs and BetaSARS-CoV-2 specific human mAbs IC50 titers against pseudovirus strains Victoria, Omicron BA.1, Omicron BA.1.1 and Omicron BA.2.

[0566]

[0567]

[0568] Table 16 - Commercial mAbs against pseudovirus strains Victoria, Omicron BA.1, Omicron BA.1.1, Omicron IC50 titers of BA.2 and Omicron BA.3.

[0569]

[0570]

[0571] Table 17-Omicron Antibody Properties

[0572]

[0573]

[0574] Table 18-22 Omicron SARS-CoV-2 specific human mAbs or commercial mAbs against various SARS-CoV-2 strains IC50 titer.

[0575]

[0576]

[0577]

[0578] Table 19 - X-ray data collection and structure refinement statistics for BA.1RBD / Omi-12-Beta54 and Omi-12 Fab

[0579]

[0580] a Omi12 is glycosylated at N102 of the heavy chain.

[0581] b The values ​​in brackets are for the highest resolution shell.

[0582] c Only rigid bodies and group B factors are refined.

[0583] Table 20 - Pseudovirus assay comparing BA.4 neutralization with that of BA.1, BA.1.1, BA.2 and BA.3

[0584]

[0585]

[0586] Table 21 - Activity of commercial antibodies against BA.4 and BA.5

[0587]

[0588] Table 22 - IC50 of BA.1 mAb against PV BA.2.75 and BA.2+N460K

[0589]

[0590] Table 23 - IC50 of commercial mAbs against PV BA.2.75

[0591]

[0592]

[0593] Table 24

[0594]

[0595] Table 25: X-ray data collection and structure refinement statistics for BA.2.75RBD / ACE2

[0596]

[0597] a The values ​​in brackets are for the highest resolution shell.

[0598] Table 26. IC50 values ​​of Omicron mAbs

[0599]

[0600] Table 27. Primer sequences used to generate pseudoviruses. Related to plasmid construction and pseudotyped lentiviral particle production.

[0601]

[0602] Table 28. X-ray data collection and structure refinement statistics. a Values ​​in parentheses are for the highest resolution shell.

[0603]

[0604]

[0605] Table 29. IC50 values ​​of Omicron mAbs and commercial monoclonal antibodies

[0606] a

[0607]

[0608] b

[0609]

[0610] Table 30. Primer sequences used to generate pseudoviruses. Related to plasmid construction and pseudotyped lentiviral particle production.

[0611]

[0612] Table 31. Table of SARS-CoV-2 lineages and genomic mutations

[0613]

[0614]

[0615] Table 32. IC50 values ​​of BA.1 mAb and commercial mAbs

[0616] a

[0617]

[0618] b

[0619]

[0620] Sequence Listing

[0621] Amino acid sequences of the heavy and light chain variable regions of the selected antibodies

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628] Nucleotide sequences of the heavy and light chain variable regions of selected antibodies

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642] Amino acid sequences of CDRs

[0643]

[0644]

[0645]

[0646]

[0647]

[0648] Amino acid sequences of the heavy and light chain variable regions of the selected antibodies

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655] Nucleotide sequences of the heavy and light chain variable regions of the selected antibodies

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665] Amino acid sequences of CDRs

[0666]

[0667]

[0668]

[0669]

[0670] Amino acid sequences of the heavy and light chain variable regions of the selected antibodies

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677] Nucleotide sequences of the heavy and light chain variable regions of selected antibodies

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687] Amino acid sequences of CDRs

[0688]

[0689]

[0690]

[0691]

[0692] SEQ ID NO:961 - Amino acid sequence encoded by IGHV1-58 germline V gene sequence MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAA

[0693] SEQ ID NO: 962 - AZD8895 (COV2-2196) heavy chain variable region nucleotide sequence

[0694]

[0695] SEQ ID NO: 963 - AZD8895 (COV2-2196) heavy chain variable region amino acid sequence:

[0696]

[0697]

[0698] SEQ ID NO: 964 - AZD8895 (COV2-2196) light chain variable region nucleotide sequence:

[0699]

[0700] SEQ ID NO: 965-AZD8895 (COV2-2196) light chain variable domain amino acid sequence:

[0701]

[0702] SEQ ID NO: 966 - spike glycoprotein amino acid sequence of WIV04 isolate Genbank Ref. QHR63260.2

[0703]

[0704]

[0705] SEQ ID NO:967—Amino acid sequence encoded by germline IGLV Kappa 3-20

[0706]

Claims

1. An antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, wherein the antibody comprises at least three CDRs of antibody Omi12 or any of the 27 antibodies in Table 3.

2. The antibody according to claim 1, comprising: (a) at least four, five or all six CDRs of an antibody in Table 3; (b) a heavy chain variable domain, the heavy chain variable domain comprising An amino acid sequence having at least 80% sequence identity to the heavy chain variable domain of an antibody in Table 3 or consisting of such an amino acid sequence; (c) a light chain variable domain comprising or consisting of an amino acid sequence having at least 80% sequence identity with a light chain variable domain of an antibody in Table 3; and / or (d) a heavy chain variable domain and a light chain variable domain, which comprise or consist of an amino acid sequence that is at least 80% identical to the heavy chain variable domain and the light chain variable domain of an antibody in Table 3, respectively.

3. The antibody according to claim 1 or claim 2, wherein the antibody in Table 3 is selected from the group consisting of: (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, Omi 23, Omi28 and Omi08; or (e) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38.

4. The antibody of claim 1, comprising CDRH1, CDRH2 and CDRH3 from a first antibody in any one of Tables 1 to 3, and CDRL1, CDRL2 and CDRL3 from a second antibody in any one of Tables 1 to 3, with the proviso that the first antibody and the second antibody are different.

5. The antibody according to claim 4, comprising a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence, wherein the heavy chain variable domain amino acid sequence has at least 80% sequence identity with the heavy chain variable domain of the first antibody from any one of Tables 1 to 3, and the light chain variable domain amino acid sequence has at least 80% sequence identity with the light chain variable domain of the second antibody from any one of Tables 1 to 3.

6. The antibody of claim 4 or claim 5, wherein the first antibody and the second antibody are derived from the same germline heavy chain v-region, optionally wherein 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.

7. The antibody according to any one of claims 4 to 6, wherein the first antibody and the second antibody are both selected from the group consisting of: (a) Omi03, Omi18, Omi29, Beta-27, Antibody 150, Antibody 158, Antibody 175, Antibody 222 and Antibody 269; optionally wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 4; (b) 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; optionally wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 5; (c) Omi12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253 and Antibody 318; optionally wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 6; (d) Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34 and Omi38; optionally wherein the antibody comprises the heavy chain variable domain and the light chain variable domain of one of the antibodies listed in Table 7; (e) Beta-22, Beta-29, Antibody 159 and Omi09; optionally wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 8; (f) Beta-20, Beta-43, Omi32 and Omi33; optionally wherein the antibody comprises the heavy chain variable domain and the light chain variable domain of one of the antibodies listed in Table 9; (g) antibodies 278, Beta-44, Omi26 and Omi41; optionally wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 10; (h) antibody 58, Omi25, Omi35 and Omi42; optionally, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of one of the antibodies listed in Table 11; or (i) Beta-56 and Omi23; optionally wherein the antibody comprises the heavy chain variable domain and the light chain variable domain of one of the antibodies listed in Table 12.

8. The antibody according to any one of the preceding claims, wherein the antibody in Table 3 to Table 12 is selected from the group consisting of: (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, Omi 23, Omi28 and Omi08; or (e) Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38.

9. The antibody according to any one of the preceding claims, which is a full length antibody, for example comprising an IgG1 constant region, or comprises an Fc region comprising at least one modification such that the serum half-life is extended.

10. The antibody according to any one of the preceding claims, wherein the antibody is derived from a germline heavy chain IGHV1-58 such as Omi-12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253 and Antibody 318, and comprises a proline at position 53 in the heavy chain variable region.

11. An antibody combination comprising two or more antibodies according to any one of the preceding claims.

12. An antibody combination, the combination comprising: (a) the antibody according to any one of claims 1 to 10; and (b) an antibody comprising at least three CDRs of an antibody in Table 1 or Table 2, for example, the antibody comprises: (i) at least four, five or all six CDRs of an antibody 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 a heavy chain variable domain of an 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 a light chain variable domain of an antibody in Table 1 or Table 2; and / or (iv) a heavy chain variable domain and a light chain variable domain, which comprise or consist of an amino acid sequence that is at least 80% identical to the heavy chain variable domain and the light chain variable domain, respectively, of an antibody in Table 1 or Table 2.

13. An antibody combination according to claim 11 or claim 12, comprising two, three or four antibodies according to any one of claims 1 to 10.

14. One or more polynucleotides encoding the antibodies according to any one of claims 1 to 10, one or more vectors comprising said polynucleotides or a host cell comprising said vectors.

15. A method for producing antibodies capable of binding to the spike protein of the coronavirus SARS-CoV-2, the method comprising culturing the host cell according to claim 14 and isolating the antibody from the culture.

16. A pharmaceutical composition comprising: (a) an antibody according to any one of claims 1 to 12 or an antibody combination according to any one of claims 11 to 13, and (b) at least one pharmaceutically acceptable diluent or carrier.

17. An antibody according to any one of claims 1 to 10, a combination according to any one of claims 11 to 13 or a pharmaceutical composition according to claim 16 for use in a method of treatment of the human or animal body by therapy.

18. An antibody according to any one of claims 1 to 10, a combination according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16, for use in a method for treating or preventing a coronavirus infection or a disease or complication associated with a coronavirus infection.

19. A method for treating or preventing a coronavirus infection or a disease or complication associated with a coronavirus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 10, a combination according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16.

20. The method of claim 19 or 20, wherein the method is used to treat SARS-CoV-2 infection, or a disease or complication associated therewith, such as COVID-19.

21. A method for identifying the presence of a coronavirus or a protein fragment thereof in a sample, the method comprising: (i) contacting the sample with an antibody according to any one of claims 1 to 10 or a combination according to any one of claims 11 to 13, and (ii) detecting the presence or absence of an antibody-antigen complex, The presence of the antibody-antigen complex indicates the presence of coronavirus or a fragment thereof in the sample.

22. A method for treating or preventing a coronavirus infection or a disease or complication associated with a subject, the method comprising identifying the presence of a coronavirus in a sample according to the method of claim 21, and treating the subject with an antibody or combination, an antiviral drug or an anti-inflammatory agent according to any one of claims 1 to 13.

23. Use of an antibody according to any one of claims 1 to 10, a combination according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 for preventing, treating and / or diagnosing coronavirus infection or a disease or complication associated therewith.

24. Use of an antibody according to any one of claims 1 to 10, a combination according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for treating or preventing coronavirus infection or a disease or complication associated therewith.

25. The antibody for use according to claim 17 or 18, the method according to any one of claims 19 to 22, and the use according to claim 23 or 24, wherein the coronavirus infection is caused by a SARS-CoV-2 strain of lineage alpha, beta, gamma, delta or omicron, optionally wherein the lineage of the omicron strain is omicronBA.1, omicron BA1.1, omicron BA.2 or omicron BA.

3.

26. An antibody derived from the germline heavy chain IGHV1-58, which is capable of binding to the spike protein of the coronavirus SARS-CoV-2, wherein the amino acid at position 58 in the heavy chain variable region according to IMGT numbering is proline or is substituted with proline.

27. The antibody of claim 26, wherein the antibody derived from germline heavy chain IGHV1-58 is AZD8895, Omi-12, Beta-47, Beta-25, Antibody 55, Antibody 165, Antibody 253 or Antibody 318.

28. An antibody capable of binding to the spike protein of the coronavirus SARS-CoV-2, the antibody comprising a heavy chain variable domain comprising an amino acid sequence having ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO:961, provided that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline.

29. The antibody of any one of claims 26 to 28, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963, provided that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline.

30. The antibody of any one of claims 26 to 29, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 591, 461, 62, 182, 262 or 332, wherein the valine at position 58 according to IMGT numbering is substituted with proline.

31. The antibody of any one of claims 26 to 29, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 961, wherein the isoleucine at position 58 according to IMGT numbering is substituted with proline.

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