Antibodies capable of binding to the spike protein of the coronavirus SARS-CoV-2

Abstract

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

Description

[Technical field]

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

[0002] The viral severe acute respiratory syndrome named COVID-19 was first reported in Wuhan, China in December 2019. The virus spread rapidly throughout the world, resulting in a pandemic with over 200 million confirmed infections and over 44 million deaths within 12 months. The pathogen, SARS-CoV-2, is a betacoronavirus related to SARS-CoV-1 and MERS coronaviruses, which all cause severe respiratory syndromes.

[0003] Within months of the identification of SARS-CoV-2 as the causative agent of COVID-19, considerable progress has been made in understanding the disease and the virus. Currently, there are several proven 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 (Non-Patent Document 1). Despite these advances, the pandemic is by no means under control, and waves of infection continue.

[0004] Coronaviruses have four structural proteins: nucleocapsid, envelope, membrane and spike (S) protein. The spike protein is the most prominent surface protein. It has an elongated trimeric structure and is responsible for target cell engagement and triggering the fusion of viral and host membranes. 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 several tissues, including epithelial cells of the upper and lower respiratory tract.

[0005] The S protein consists of two subunits, S1, which mediates receptor binding, and S2, responsible for fusion of viral and host cell membranes. The S protein is a dynamic structure that can transition to a post-fusion state upon receptor binding or cleavage between S1 and S2 following trypsinization. In some SARS-CoV-2 sequences, a furin protease cleavage site is inserted between the S1 and S2 subunits, and mutation of the cleavage site mitigates disease in animal models. The S1 fragment occupies the membrane-distal tip of S and can be subdivided into an N-terminal domain (NTD) and a receptor-binding domain (RBD). Both regions are immunogenic, while the RBD contains the interaction surface for ACE2 binding. Although normally packed against the top of S2, the RBD can pivot upwards to engage ACE2. Monoclonal antibodies (mAbs) recognize one or both of the "up" and "down" conformations.

[0006] The S protein is relatively conserved between SARS-CoV-2 and SARS-CoV-1 (76%), while the RBD and NTD are less conserved (74% and 50%, respectively) than the S2 domain (90%). Conservation with 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.

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

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

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

[0010] Omicron BA.2 was described at approximately the same time as BA.1. The proportion of Omicron infections caused by BA.2 is increasing in several countries and has become the predominant sublineage in Denmark and India.

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

[0012] BA.2 appears to have a slight transmission advantage over BA.1, with no evidence of increased disease severity. In early April 2022, two new Omicron lineages were reported from Gauteng province in South Africa, named BA.4 and BA.5. BA.4 and BA.5 (with identical S sequences) became the predominant Omicron strains in Gauteng and fueled a new wave of infections in South Africa.

[0013] Since June 2022, BA.4 / 5 (Non-Patent Document 7), which has higher receptor binding affinity and significantly enhanced escape from antibody responses, has spread rapidly from South Africa around the world and is now the new global dominant strain, with BA.5 dominating in many regions. These variants (especially BA.5) now account for the majority of sequenced cases in many countries.

[0014] In early May 2022, a new Omicron sublineage, designated BA.2.75, emerged in India. This strain has since spread to many countries, including the UK, the US, Australia, Germany and Canada. However, the true prevalence of BA.2.75 is difficult to determine because sequencing is incomplete and significantly reduced in many countries.

[0015] All of these variants contain multiple mutations within S, including alterations in the RBD, NTD, and possibly 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 within or closely adjacent to the ACE2 interacting surface, where the RBD mutations have the potential to modulate ACE2 interaction and impair binding of neutralizing antibodies. Increased affinity of ACE2 interaction is prominent for alpha, beta, gamma, and delta (7-, 19-, 19-, and 2-fold, respectively), which may play a role in enhancing viral transmissibility. Omicron contains an unprecedented number of mutations concentrated in the spike (S) gene, with 30 substitutions of residues, and 6 deletions and 3 insertions. Omicron BA.1 (RBD mutations of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H) contains the unique mutations S371L, G446S, and G496S, and in some isolates R346K (BA.1.1), while BA.2 contains S371F, T376A, D405N, and R408S. BA.3 does not contain the unique mutations associated with BA.1 and BA.2 and appears to be a fusion of the two, resembling BA.1 at the N-terminus and switching from the mutation G496S to one resembling BA.2 at the C-terminus.

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

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

[0018] Considering the physicochemical properties of the side chain of residue 452, BA.2.13 is predicted to be a relatively modest change, going from L to M, which increases the size of the side chain but maintains hydrophobicity. BA.2.12.1 going from L to Q introduces some polar character, while BA.2.11 is the most radical, going from L to R, which introduces a large basic amino acid.

[0019] Further variants BA.4 and BA.5, which have identical S sequences, are believed 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. In particular, residues 69 and 70 of the NTD are deleted (also found in Alpha, BA.1, BA.3), and 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, similar to the Victoria / Wuhan strain. Looking at the RBD, BA.4 and BA.5 contain mutations at all previously mentioned positions in VoC alpha (N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N501Y), and delta (L452, T478K), with the only difference being E484A instead of E484K in beta and gamma in BA.4 and BA.5.

[0020] As of September 2022, a new variant related to BA.4 / 5, designated BA.4.6, has emerged and spread in the United States, where BA.5 predominates (prevalence is 87.5% as of September 10, 2022, a three-fold increase 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 R346K mutation in BA.1.1 reduces serum neutralization compared to BA.1 and impairs the activity of some monoclonal antibodies (mAbs), raising concerns that the R346T mutation may enhance antibody evasion over BA.4 / 5 (Non-Patent Document 5). SARS-CoV-2 detection kits using monoclonal antibodies have also been developed. Examples include lateral flow tests, such as those by Innova (SARS-CoV-2 Antigen Rapid Qualitative Test) and Quidel (Sofia 2 SARS Antigen FIA). However, these tests have been reported to be highly inaccurate.

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

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

[0023] Following BA.5, several new trends were observed in the evolution of Omicron: i) the emergence of "second generation" BA.2 variants (including derived forms of BA.5) - variants with long lineage branches, multiple antigenic mutations, and lack of genetic intermediates, e.g., BA.2.75, BJ.1, BS.1, BA.2.10.4, and BA.2.3.20 (Non-Patent Document 12), and ii) accelerated antigenic drift seen in both BA.5 (Non-Patent Document 7) and 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) generated XBB. Many of these variants show significant convergent evolution at known antigenic RBD residues, and the mutations are in regions that may compromise binding of neutralizing antibodies, resulting in further escape from protection against infection conferred by vaccines or previous SARS-CoV-2 infection, including previous omicron infection.

[0024] Currently, several lineages are growing rapidly from within both the BA.2 and BA.5 branches. Most notable is the large degree of convergent evolution, particularly 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 reversion 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 the BA.2.75 branch (naturally containing G446S, N460K and the reversion R493Q), BA.2.75.2 (R346T and F486V), BN.1 (R346T, K356T, F490S). Examples of several other second generation BA.2 variant strains include 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), BS.1 (BA.2.3.2.1; R346T, L452R, V445P, G446S, V483A, and F490V), and BA.2.10.5 (G446S, F486P, S494P, and R493Q reversion). , N460K, G476S), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and Q493R reversion) and finally the BA.2.75xBJ.1 recombinant, XBB (containing R346T, L368I, V445P, G446S, N460K, F486S, F490S relative to BA.2).

[0025] These second-generation BA.2 variants have become dominant globally, 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 spreading rapidly in North America.

[0026] Outside the RBD, convergent evolution is less frequent but still present. Many second generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in 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).

[0027] All currently licensed SARS-CoV-2 vaccines are designed to induce antibody (and T cell) responses against S and contain the S sequence found in the original Wuhan strain. Thus, particular concerns exist regarding whether S mutations within VoC could cause immune evasion and contribute to vaccine failure or susceptibility to repeat infection in previously infected individuals.

[0028] The extensive mutational burden of Omicron S inhibits the activity of most mAbs that bind to three of these potent antibody binding sites, the ACE-2 footprint, the N343 glycan surroundings, and the NTD, resulting in a severely reduced or complete loss of neutralizing capacity of sera from natural infection or vaccination, contributing to the increased transmissibility and explosive spread of Omicron S. [Prior art documents] [Non-patent literature]

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

[0030] The object of the present invention is to identify further improved antibodies useful for preventing, treating and / or diagnosing coronavirus infections and diseases and / or complications associated with coronavirus infections, including COVID-19, in particular omicron variants of concern (VoC) and as yet unidentified variants with additional mutations in the ACE-2 footprint, RBD and / or NTD within the spike protein of SARS-CoV-2. [Means for solving the problem]

[0031] We 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 of the antibodies in Table 3 showed broadly effective and potent neutralizing effects against the hCoV-19 / Wuhan / WIV04 / 2019 strain as well as SARS-CoV-2 strains of various lineages, including Victoria (Wuhan+S247R), alpha, beta, gamma, delta, and 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 XBB and / or Omicron XBB.1).

[0032] Many of the mAbs in Table 3 used public V genes (V genes shared by a large population). We have previously shown that it is possible to generate additional antibodies by swapping the light and heavy chains of antibodies in Tables 1, 2 and 3 that are derived from the same public V gene. Antibodies derived from the same public V gene provided particularly useful mixed chain antibodies.

[0033] In particular, the inventors found that antibodies Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42 are particularly effective at cross-neutralizing SARS-CoV-2 strains Victoria, alpha, beta, gamma, delta and omicron.

[0034] Thus, the present invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, the antibody comprising 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 coronavirus SARS-CoV-2, the antibody comprising at least three CDRs of antibody Omi12 or any one of the 27 antibodies in Table 3.

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

[0037] The invention also provides antibody combinations comprising: (a) an antibody of the 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 to the 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 to the 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 comprising, or consisting of, amino acid sequences having at least 80% identity to the heavy chain variable domain and light chain domain, respectively, of an antibody in Table 1 or Table 2.

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

[0039] The invention also provides a method for producing antibodies capable of binding to the spike protein of coronavirus SARS-CoV-2, the method comprising culturing a host cell of the 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 invention, and (b) at least one pharma- ceutically acceptable diluent or carrier.

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

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

[0043] The present invention also provides 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, antibody combination, or pharmaceutical composition of the present invention.

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

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

[0046] The present invention also provides the use of an antibody, antibody combination or pharmaceutical composition of the invention for preventing, treating and / or diagnosing a coronavirus infection or a disease or complication associated therewith.

[0047] The invention also provides the use of an antibody, antibody combination or pharmaceutical composition of the invention for the manufacture of a medicament for treating or preventing a coronavirus infection or a disease or complication associated therewith. [Brief description of the drawings]

[0048] [Figure 1] Generation of the Omicron BA.2 sublineage and Omicron mAb panel. Figure 1 only relates to the first 22 Omicron antibodies (i.e. Omi02-Omi35) disclosed in Tables 13 and 14. (A) FRNT50 titers against Victoria and Omicron BA.1 from donors for the production of Omicron mAbs are shown. (B) FACS plot showing sorting of B cells using full-length Omicron S. (C) Percentage of RBD and NTD binding antibodies found in Omicron mAbs compared to pandemic early mAbs. (D) Heavy and light chain variable gene usage. (E) Somatic mutations found in potent Omicron mAbs (FRNT50<100ng / ml) compared to pandemic early set. [Diagram 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. [Diagram 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 with Victoria, alpha, beta, gamma, delta, and omicron viruses using sera obtained <14 days and >21 days after symptom onset, (D) Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 pseudoviruses with early and late sera. Geometric mean titers are shown above each column. Wilcoxon matched-pairs signed-rank test (A and B) and Mann-Whitney test (C and D) were used for analysis to calculate two-sided P values. [Figure 4] Pseudovirus neutralization curves. Early pandemic mAbs (B) Pseudovirus neutralization curves for BA.1, BA.1.1, BA.2, and BA.3 with beta mAbs. [Diagram 5] Figure 3. (A) Viral neutralization titers shown for live virus, (B) pseudovirus. Geometric mean titers are shown above each column. Two-sided P values ​​were calculated using Wilcoxon matched-pairs signed rank test for analysis. (C) Pseudovirus neutralization curves of selected VH1-58mAbs and control VH3-53mAb222 against Victoria and Iota(S477N). [Figure 6] Structure of BA.1 RBD with Omi-12 Fab. (A) Two ternary complexes of Omi-12 and beta-54 Fab and 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 low-resolution ternary complex density) are compared by superimposing the RBDs. The Fab of one complex is light colored (HC red, LC blue in schematic) and the Fab of the other complex is light colored. (B) Binding mode of Omi-12. (C) Zoomed-in view of the binding difference of Omi-12 with Fab253 in complex with pandemic early RBD (light blue) and beta-47 in complex with beta RBD (light cyan). (D) Somatic mutation V53P contributes to refolding of the H3 loop so that Q493R can be accommodated by Omi-12. [Figure 7] Pseudovirus neutralization assays of BA.4 / 5 with vaccine and BA.1 immune sera. IC50 values ​​for the indicated viruses using sera obtained from vaccine recipients 28 days after the third dose of vaccine (A) AstraZeneca AZD AZD1222 (n=41) and (B) 4 weeks after the third dose of Pfizer BNT162b2 (n=20). Sera were collected from breakthrough BA.1 infected volunteers at (C) early, ≤14 days (n=12) from symptom onset (median 13 days) and (D) late, ≥21 days (median 38 days) from symptom onset, n=16. Comparisons were made for neutralization titers against Victoria (early pandemic strain), BA.1, BA.1.1, BA.2 and BA.3. Geometric mean titers are shown above each column. Wilcoxon matched-pairs signed rank test was used for analysis to calculate two-sided P values. [Figure 8] Pseudovirus neutralization assays for Omicron and commercial monoclonal antibodies. Neutralization curves for a panel of 28 monoclonal antibodies generated from samples taken from vaccinees infected with BA.1. The titration curve for BA.1 is compared to BA.1, BA.1.1, BA.2 and BA.3. mAbs putatively affected by L452R and F486L are indicated. [Figure 9] Omicron sublineages compared to BA.4 / 5. (A) Comparison of S protein mutations in Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 / 5 with NTD and RBD boundaries indicated. (B) Location of RBD mutations (gray surface, ACE2 footprint in dark green). Mutations common to all Omicron lineages are shown in white (Q493R, reverted in BA.4 / 5, is shown as 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 clear stick on the surface. [Figure 10]Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs. (A) Binding of BA.4 / 5 RBD was greatly reduced compared to that of BA.2, and binding could not be accurately measured as indicated by a single injection of 200 nM RBD into a sample flow cell containing IgG Omi-31. (B-C; E-I) Sensorgrams showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red: original binding curve, black: fitted curve). Kinetic data are also shown. (D) Measurement of the affinity of BA.4 / 5 RBD with Omi-12 using 1:1 binding equilibrium analysis. [Figure 11] Interactions between mAbs and the BA.4 / 5 mutant sites. Overall structures of (A) BA.1-RBD / Omi-31 (PDB 7ZFB), (B) BA.1-RBD / Omi-32 (PDB 7ZFE), (C) BA.1-RBD / Omi-25 (PDB 7ZFD), (D) BA.1-RBD / Omi-42 (PDB 7ZR7), (E) Wuhan-RBD / AZD8895 (PDB7L7D), and (F) BA.1-RBD / Omi-3 (PDB 7ZF3) complexes (left panel) and their interactions with the BA.4 / 5 mutant sites (≦4 Å) (right panel). In the left panel, the RBD is shown as a surface representation, the BA.4 / 5 mutation site is highlighted in magenta, two additional mutation sites at 452 and 486 in BA.4 / 5 are highlighted in cyan, the FabLC is blue, and the HC is shown as red ribbons. In the right panel, the RBD, Fab HC, and LC side chains are depicted as grey, red, and blue sticks, respectively. In (B), L452R (green stick) is modeled to show that a salt bridge to D99 in CDR-H3 can be formed (yellow dashed line). (D) Beta-RBD / Omi-42 complex showing that the Fab is not in contact with either of the two BA.4 / 5 mutation sites. [Figure 12]ACE2 RBD affinity. (A)-(D) SPR sensorgrams comparing ACE2 binding of BA.4 / 5 RBD (A) to ancestral (Wuhan) (B), BA.1 (C) and BA.2 RBD (D). Wuhan, BA.1 and BA.2 data have been previously reported (Nutalai et al., 2022). (E)-(G) Electrostatic surfaces, (E) from left to right, respectively, early pandemic, delta and BA.1 RBD, (F) open view of BA.2 RBD and ACE2 in the BA.2 RBD / ACE2 complex (PDB7ZF7), and (G) BA.4 / 5 RBD (modeled based on the structure of BA.2 RBD). Diamonds in ACE2 and RBD indicate interaction regions. [Figure 13] Antigen mapping. (A) Neutralization data and model (log titer values) used to calculate the antigen map in (B). Columns represent sera taken from vaccinated volunteers or infected patients. Rows are challenge strains: Victoria, alpha, delta, beta, gamma, BA.1, BA1.1, BA.2, BA.3, BA.4 / 5 in order. Values ​​are colored according to deviation from a reference value, which is calculated based on serotype as the average of neutralization titers from the row giving the highest value. (B) Orthogonal view of the antigen map showing BA.4 / 5 in relation to previous VoC and 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 drop in neutralization titer when one of the corresponding strains is challenged with serum obtained from infection with the other. Figure 6. ACE2 / RBD affinity and antigen mapping [Figure 14] Neutralization curves for VH1-58 mAb. Pseudovirus neutralization curves for early pandemic mAb 253 (Dejnirattisai et al., 2021a) and Beta-47 (Liu et al., 2021b) against a panel of Victoria and Omicron lineage constructs. [Figure 15]Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs. (A-F) Sensorgrams showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red: original binding curves, black: fitted curves). Kinetic data are also shown. (G-K) Binding of BA.4 / 5 RBD was significantly reduced compared to that of BA.2, and the binding shown could not be accurately measured by a single injection of 200 nM RBD into a sample flow cell containing the indicated mAb. [Figure 16] Sequence changes in 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. NTD and RBD boundaries are marked. (B) Surface representation of mutated residues in the BA.2.75 RBD compared to the BA.2 RBD. The positions of the BA.2 RBD mutations (gray surface, ACE2 footprint dark green) are indicated, and the residues mutated in BA.2.75 are shown in orange and labeled. [Figure 17] Pseudovirus neutralization assays of BA.2.75 with vaccine and BA.1 and BA.2 immune sera. IC50 values ​​for the indicated viruses using sera 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) Sera from volunteers with vaccine breakthrough BA.1 (n=16) or BA.2 (n=23) infection. (EC) IC50 values ​​of single RBD point mutations inserted into BA.2 pseudovirus using Pfizer BNT162b2 sera (n=22). Geometric mean titers are shown above each column. Two-sided P values ​​were calculated using Wilcoxon matched-pairs signed rank test for analysis. [Figure 18]ACE2 / RBD affinity. SPR sensorgrams showing ACE2 binding of BA.2.75 RBD using ACE2-Fc (A) or biotinylated ACE2 (B) as ligand, compared to the binding of BA.2 (C), BA.4 / 5 (D), alpha (E) and BA.2+R493Q (F) to the 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. [Figure 19] Pseudovirus neutralization assay for monoclonal antibodies. (A) Neutralization curves for a panel of 28 mAbs generated from samples taken from vaccine recipients infected with BA.1. The titration curve for BA.2.75 is compared to Victoria, BA.1, BA.1.1, BA.2 and BA.4 / 5. IC50 titers are shown in Table 22. (B) Pseudovirus neutralization assay with mAbs developed for humans. IC50 titers are shown in Table 23. Data for Victoria, BA.1, BA.1.1 and BA.2 and BA.4 / 5 were used for comparison and were taken from Tuekprakhon et al., 2022. [Figure 20] Structure of the BA.2.75 RBD / ACE2 complex. (A) Overall structure of the BA.2.75 RBD / ACE2 complex. ACE2 is shown as a green ribbon, the RBD is shown on the surface, common mutations in BA.2 are highlighted in magenta, and distinct mutations are highlighted in orange. (B) The interface of BA.2.75 RBD (gray) and ACE2 (green) compared to the interfaces of BA.2 and ACE2 (both salmon colored). A close-up shows the interaction of Q496R and Q493 (R493 in BA.2) with ACE2. [Figure 21]Interaction between mAb and BA.75 mutation sites. (A) Front and back view of the binding mode of Omi-3 (PDB, 7ZF3) and Omi-18 (PDB, 7ZFC) in complex with Omicron BA.1 RBD by superimposing the RBDs. The RBDs are shown as grey surface representations, mutations common to both BA.2 and BA.2.75 are shown in magenta, and the four mutations that differ between the two are shown in cyan. VH and VL are shown as ribbons, with Omi-3 shown in red and blue, and Omi-18 in light blue and salmon, respectively. (B) Interaction between N460 of the RBD and CDR-H2 of the Fab. (C) Contact between R493 of the RBD and CDR-H3 of the Fab. In (B) and (C), the RBD associated with Omi-3 is shown in grey, Omi-18 in cyan, and the Fab colors are the same as in (A). (D) AZD1061 binds to the ancestral SARS-CoV-2 RBD (PDB, 7L7E), contacting (E) G446 of the RBD and CDR-L2 of the Fab. (E) AZD8895 binds to the ancestral SARS-CoV-2 spike RBD (PDB, 7L7E), contacting (F) Q493 of the RBD and CDR-H2 of the Fab. In (D)-(F), the RBDs are depicted and colored as in (A), with HCs in red and LCs in blue. [Figure 22] Antigenic mapping. (A) Orthogonal view of the antigenic map showing BA.2.75 in relation to previous VoC and BA.1, BA.1.1, BA.1 and BA.2 positions calculated from pseudovirus neutralization data. The distance between two positions is proportional to the drop in neutralization titer when one of the corresponding strains is challenged with sera obtained by infection with the other. As the figure is a projection of a three-dimensional distribution, a scale is not provided, but the variation can be calibrated by comparing (i) BA.1 to BA.2 (2.93-fold reduction) and (ii) BA.2 to BA.4 / 5 (3.03-fold reduction). (B) As in (A), but including only Omicron sublineage and early pandemic viruses to allow a more accurate projection of this subset in three dimensions. Note that the response of these viruses to all sera is included in the calculation. [Diagram 23]Pseudovirus neutralization assay for monoclonal antibodies. (A) Neutralization curves for a panel of 28 monoclonal antibodies generated from samples taken from vaccinees infected with BA.1. Titration curves of single mutations of BA.2.75 in the BA.2 backbone are compared to BA.2 and BA.2.75. IC50 titers are shown in Table 24. [Figure 24] Surface plasmon resonance (SPR) analysis of the interaction between BA.2 or BA.2.75 RBD and selected mAbs. (A) Binding of Omi-29 (IGHV3-53) to BA.2.75 RBD is significantly reduced compared to that of BA.2, as shown by a single injection of 1 μM Omi-29Fab into a sample flow cell containing biotinylated BA.2 or BA.2.75 RBD. (B) Binding of Omi-36 (IGHV3-66) to BA.2.75 RBD is significantly reduced compared to that of BA.2, as shown by a single injection of 0.2 μM BA.2 or BA.2.75 RBD into a sample flow cell containing Omi-36 in IgG form. (C-H) Sensorgrams showing the interaction between BA.2 or BA.4 / 5 RBD and selected mAbs (red / colored: original binding curves, black: fitted curves). Kinetic data are also shown. [Diagram 25] Neutralization of BA.2.75 by a panel of convalescent sera collected from historical infections with variants. Neutralization titers of the indicated sera against BA.2.75 and the indicated pseudoviruses. Data other than BA.2.75 were taken from Tuekprakhon et al., 2022. [Figure 26] Primers for site-directed PCR mutagenesis of BA.2.75 RBD. Site-directed PCR mutagenesis was performed using the BA.2 spike construct as a template. D339H, G446S, N460K and R493Q mutations were introduced using the primers shown. [Figure 27]Characterization of BA.2.11, BA.2.12.1 and BA.2.13 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 vaccination of vaccines (a) AstraZeneca AZD1222 (n=41), (b) Pfizer BNT162b2 (n=18). (c) Neutralization titers were measured for sera from vaccinated volunteers with breakthrough BA.1 infection. Comparisons were made with neutralization titers against Victoria, BA.1, BA.1.1, BA.2 and BA.4 / 5 previously reported in Tuekprakhon et al. (2022). Geometric mean titers are shown above each column. Two-tailed p-values ​​were calculated using Wilcoxon matched-pairs signed rank test for analysis. (d-g) SPR sensorgrams showing ACE2 binding of the RBDs of BA.2.11 (e), BA.2.12.1 (f) and BA.2.13 (g) compared to binding to the BA.2 RBD (h) (red: experimental binding curves, black: fitted curves). Kinetic data are also shown. Data for BA.2 RBD are reported by Nutalai et al. (2022). (h-m) Crystal structure of the BA.2.12.1 RBD / beta-27 / NbC1 complex. (h) The overall structure is shown as a Cα trace with the RBD (grey), beta-27HC (red) and LC (blue) and NbC1 (yellow). The Cαs of residues L452Q, F486, Q493R (L, F and R in BA.2, R, V and Q in BA.4 / 5) are shown as spheres. (i) Comparison of beta-27 binding modes in the BA.2.12.1 RBD / beta-27 / NbC1 (RBD surface representation, HC in red, LC in blue), BA.4 / 5 RBD / beta-27 / NbC1 (cyan, PDB 7ZXU) and beta RBD / beta-27 (green, PDB 7PS1) complexes by superimposing the RBDs. Apart from the flexible N- and C-terminal regions of the RBD, major differences occur in the N-terminus of the Fab HC, α2 helix, 371-375 loop and G446 loop of the RBD and in the CDR-H1. The CDR-L3 has a doublet structure in the BA.4 / 5 RBD complex and a single structure in the other two complexes (i).The HCN terminus and CDR-H1 contacting residue 486 of the RBD differ from those of both the beta and BA.4 / 5 RBD complexes, the latter containing the F486V mutation. This difference is likely caused by contacts from the symmetry-related C1 nanobody shown as grey bonds in (j). (k) Structural differences in the G446 loop of the BA.4 / 5 RBD are also induced by crystal contacts. (l) The 371-375 loop with the S371F, S373P and S375F mutations of 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) The mutation at 452 does not cause significant local structural changes. R452 of BA.4 / 5 has a bipartite structure. [Figure 28] Pseudovirus neutralization assays of BA.4.6 by vaccines. BA.1, BA.2, BA.4.5 immune sera (a-d) and monoclonal antibodies (e-f). IC50 values ​​for the indicated viruses using sera obtained from vaccinees 28 days after the third dose of Pfizer BNT162b2 vaccine (n=22, a). IC50 values ​​for the indicated viruses against sera from volunteers with vaccine breakthrough BA.1 (n=14, b), BA.2 (n=23, c) and BA.4 / 5 (n=11, d) infections. Geometric mean titers are shown above each column. Two-sided P values ​​were calculated using Wilcoxon matched-pairs signed rank test for analysis. Neutralization curves against BA.4.6 of a panel of 28 monoclonal antibodies generated from samples taken from vaccine recipients infected with BA.1(e) were compared to the Victoria, BA.1, BA.1.1, BA.2, BA.4 / 5 and BA.2.75 variants. Neutralization curves of a panel of 14 commercially available monoclonal antibodies against the same variants(e). IC50 values ​​are shown in Tables 29A and 29B. [Figure 29]Pseudovirus neutralization assay. Pseudovirus neutralization assay for Omicron monoclonal antibodies for Table 26 with IC50 titers shown. Neutralization curves for a panel of 27 monoclonal antibodies generated from samples taken from vaccinees infected with BA.1. Titer curves for BA.2.11, BA.2.12.1 and BA.2.13 are compared to BA.2. [Diagram 30] Surface plasmon resonance (SPR) analysis of the interaction between BA.2.12.1 or BA.2 RBD and selected mAbs (Omi-6 and Omi-31). (a) Measurement of the affinity of BA.2.12.1 RBD to Omi-6 using a 1:1 binding equilibrium analysis. (b), (c), (d) Sensorgrams showing the interaction between BA.2.12.1 or BA.2 RBD and selected mAbs (red: original binding curve, black: fitted curve). Kinetic data are also shown. [Diagram 31] Neutralization assays. Neutralization curves using lentivirus pseudotyped with the S gene of the indicated BA.2 sublineages (A) Omi-mAb, (B) commercial mAb. See also Table 32. The "BA.4+all" variant is a synthetic variant designed following evaluation of different mutations occurring in the SARS-CoV-2 Omicron S gene. These mutations were combined and incorporated into the Omicron BA.4S gene to generate an artificial S gene called "BA.4+all". This variant was created only as an experimental tool and does not occur in nature or correspond to the S gene of any circulating SARS-CoV-2 variant. [Diagram 32] Serum neutralizing IC50 titers (dilution fold) of lentivirus pseudotyped with the S gene of the indicated BA.2 sublineage. Sera obtained 28 days after (A) the third dose of BNT162b2 vaccine or infection with (B) BA.1, (C) BA.2, or (D) BA.4 / 5. Geometric mean titers are shown above each column. Two-sided P values ​​were calculated using Wilcoxon matched-pairs signed rank test (C and D) and Mann-Whitney test (E). [Diagram 33]Heatmap of antibody binding. Heatmap showing IC50 (μg / ml) of various antibodies against Victoria and Beta strains in both vaccinated and unvaccinated samples. [Diagram 34] Neutralization assays. Neutralization curves using lentiviruses pseudotyped with the S genes of the indicated BA.2 sublineages. [Diagram 35] Heatmap of IC50 neutralization titers for a 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 seen early in the pandemic are also included for comparison. Victoria, BA.2 and BA.4 / 5 live virus assay data and pseudovirus data were previously reported by Tuekprakon et al. (2022). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Antibodies of the Invention The antibodies of the present invention specifically bind to the spike protein of SAR-CoV-2, in particular to the S1 subunit of the spike protein, such as the receptor binding domain (RBD) or the N-terminal domain (NTD).

[0050] An antibody of the invention may comprise at least three CDRs of an antibody in Table 3. The antibody may comprise at least four, five or all six CDRs of an 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 to a heavy chain variable domain of an 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 to a light chain variable domain of an 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 to the heavy chain variable domain and light chain domain, respectively, of an antibody in Table 3. The antibody may be any one of the antibodies in Table 3.

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

[0052] The antibodies in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38. These antibodies were surprisingly found to retain potent neutralization of live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta, omicron (e.g., IC50≦0.1 μg / ml against all live strains tested).

[0053] 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. These antibodies were surprisingly found to retain potent neutralization of live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta, omicron (e.g., IC50≦0.05 μg / ml against all live strains tested).

[0054] The antibodies in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39 and Omi42. These antibodies were surprisingly found to retain potent neutralization of live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta, omicron (e.g., IC50≦0.02 μg / ml against all live strains tested).

[0055] The antibodies in Table 3 may be selected from the group consisting of: Omi03 and Omi12. These antibodies were surprisingly found to retain very potent neutralization of live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta, omicron (e.g., IC50≦0.01 μg / ml against all live strains tested).

[0056] The antibodies in Table 3 may be selected from the group consisting of: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42. These antibodies were surprisingly found to retain very strong neutralization of live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta, omicron.

[0057] 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, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 695, 696 and 697, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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).

[0058] The heavy chain domain of Omi03 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi03 and may not comprise a light chain of Omi03. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 695, 696 and 697, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 692.

[0059] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi03 and may not comprise a heavy chain of Omi03. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0060] In one embodiment, the antibody in Table 3 may be Omi12. Omi12 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 735, 736 and 737, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 738, 739 and 740, 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 Omi12 (i.e., SEQ ID NO: 732). 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 Omi12 (i.e., SEQ ID NO: 734). In one embodiment, an antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain, respectively, that comprise or consist 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 Omi12 (i.e., SEQ ID NOs: 732 and 734, respectively).

[0061] The heavy chain domain of Omi12 is derived from the IGHV1-58 v-region, and the inventors have previously demonstrated that heavy and light chain swapping between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi12 and may not comprise a light chain of Omi12. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences designated in SEQ ID NOs: 735, 736 and 737, respectively. An 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 Omi12 (i.e. SEQ ID NO: 732). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 732.

[0062] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi12 and may not comprise a heavy chain of Omi12. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0063] In one embodiment, the antibody in Table 3 may be Omi02. Omi02 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 685, 686 and 687, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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 Omi02 (i.e., SEQ ID NOs: 682 and 684, respectively).

[0064] The heavy chain domain of Omi02 is derived from the IGHV1-69 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi02 and may not comprise a light chain of Omi02. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 685, 686 and 687, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 682.

[0065] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi02 and may not comprise a heavy chain of Omi02. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0066] In one embodiment, the antibody in Table 3 may be Omi08. Omi08 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 715, 716 and 717, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 that respectively comprise or consist 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).

[0067] In one embodiment, the antibody in Table 3 may be Omi42. Omi42 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 955, 956 and 957, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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 Omi42 (i.e., SEQ ID NOs: 952 and 954, respectively).

[0068] The heavy chain domain of Omi42 is derived from the IGHV3-9 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi42 and may not comprise a light chain of Omi42. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 955, 956 and 957, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 952.

[0069] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi42 and may not comprise a heavy chain of Omi42. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0070] In one embodiment, the antibody in Table 3 may be Omi16, which was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 745, 746 and 747, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 748, 749 and 750, 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 Omi16 (i.e., SEQ ID NO: 742). 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 Omi16 (i.e., SEQ ID NO: 744). In one embodiment, an antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain, respectively, that comprise or consist 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 Omi16 (i.e., SEQ ID NOs: 742 and 744, respectively).

[0071] The heavy chain domain of Omi16 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi16 and may not comprise a light chain of Omi16. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences designated in SEQ ID NOs: 745, 746 and 747, respectively. An 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 Omi16 (i.e. SEQ ID NO: 742). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 742.

[0072] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi16 and may not comprise a heavy chain of Omi16. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0073] In one embodiment, the antibody in Table 3 may be Omi18. Omi18 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 765, 766 and 767, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 768, 769 and 770, respectively. In one embodiment, the 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 to the heavy chain variable domain of the antibody Omi18 (i.e., SEQ ID NO: 762). In one embodiment, the 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 to the light chain variable domain of the antibody Omi18 (i.e., SEQ ID NO: 764). In one embodiment, an antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain, respectively, that comprise or consist 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 Omi18 (i.e., SEQ ID NOs: 762 and 764, respectively).

[0074] The heavy chain domain of Omi18 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi18 and may not comprise a light chain of Omi18. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 765, 766 and 767, respectively. An antibody may comprise a heavy chain variable domain that comprises or consists 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 Omi18 (i.e. SEQ ID NO: 762).

[0075] The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO:762.

[0076] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi18 and may not comprise a heavy chain of Omi18. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0077] In one embodiment, the antibody in Table 3 may be Omi20. Omi20 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 775, 776 and 777, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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 Omi20 (i.e., SEQ ID NOs: 772 and 774, respectively).

[0078] The heavy chain domain of Omi20 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful in the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi20 and may not comprise a light chain of Omi20. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 775, 776 and 777, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 772.

[0079] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi20 and may not comprise a heavy chain of Omi20. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0080] In one embodiment, the antibody in Table 3 may be Omi23. Omi23 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 785, 786 and 787, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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 Omi23 (i.e., SEQ ID NOs: 782 and 784, respectively).

[0081] The heavy chain domain of Omi23 is derived from the IGHV4-31 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi23 and may not comprise a light chain of Omi23. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 785, 786 and 787, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 782.

[0082] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi23 and may not comprise a heavy chain of Omi23. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0083] In one embodiment, the antibody in Table 3 may be Omi28. Omi28 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 835, 836 and 837, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, the antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain that respectively comprise or consist of an amino acid sequence having ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, 100% sequence identity to the heavy chain variable domain and the light chain variable domain of the 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 shuffling of heavy and light chains between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, the antibody of the present invention may comprise the heavy chain of Omi28 and may not comprise the light chain of Omi28. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified 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.

[0084] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi28 and may not comprise a heavy chain of Omi28. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0085] In one embodiment, the antibody in Table 3 may be Omi39. Omi39 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 935, 936 and 937, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 that respectively comprise or consist 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 Omi39 (i.e., SEQ ID NOs: 932 and 934, respectively).

[0086] In one embodiment, the antibody in Table 3 may be Omi17. Omi17 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 755, 756 and 757, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 758, 759 and 760, 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 Omi17 (i.e., SEQ ID NO: 752). 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 Omi17 (i.e., SEQ ID NO: 754). In one embodiment, an antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain, respectively, that comprise or consist 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 Omi17 (i.e., SEQ ID NOs: 752 and 754, respectively).

[0087] The heavy chain domain of Omi17 is derived from the IGHV3-66 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi17 and may not comprise a light chain of Omi17. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences designated in SEQ ID NOs: 755, 756 and 757, respectively. An 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 Omi17 (i.e. SEQ ID NO: 752). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 752.

[0088] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi17 and may not comprise a heavy chain of Omi17. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0089] In one embodiment, the antibody in Table 3 may be Omi29. Omi29 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 845, 846 and 847, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 that respectively comprise or consist 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 Omi29 (i.e., SEQ ID NOs: 842 and 844, respectively).

[0090] The heavy chain domain of Omi29 is derived from the IGHV3-53 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi29 and may not comprise a light chain of Omi29. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 845, 846 and 847, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 842.

[0091] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi29 and may not comprise a heavy chain of Omi29. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0092] In one embodiment, the antibody in Table 3 may be Omi36, which was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 915, 916 and 917, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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).

[0093] In one embodiment, the antibody of the present invention may comprise a heavy chain variable domain and a light chain variable domain that respectively comprise or consist of an amino acid sequence having ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, 100% sequence identity to the heavy chain variable domain and the light chain variable domain of the 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 heavy and light chain swapping between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, the antibody of the present invention may comprise the heavy chain of Omi36 and may not comprise the light chain of Omi36. For example, the antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified 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.

[0094] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi36 and may not comprise a heavy chain of Omi36. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0095] In one embodiment, the antibody in Table 3 may be Omi38. Omi38 was found to neutralize live SARS-CoV-2 variant strains Victoria, alpha, beta, gamma, delta and Omicron, as well as pseudoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.3 (see Tables 13 and 14, FIG. 2). In one embodiment, the antibody of the invention may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 925, 926 and 927, respectively, and CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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, respectively, that comprise or consist 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 Omi38 (i.e., SEQ ID NOs: 922 and 924, respectively).

[0096] The heavy chain domain of Omi38 is derived from the IGHV1-69 v-region, and the inventors have previously demonstrated that heavy and light chain shuffling between antibodies derived from the same v-region results in antibodies that are particularly useful for the present invention (as further described below). Thus, an antibody of the present invention may comprise a heavy chain of Omi38 and may not comprise a light chain of Omi38. For example, an antibody may comprise CDRH1, CDRH2 and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 925, 926 and 927, respectively. An 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). An antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 922.

[0097] Alternatively, in one embodiment of the invention, the antibody may comprise a light chain of Omi38 and may not comprise a heavy chain of Omi38. For example, the antibody may comprise CDRL1, CDRL2 and CDRL3 having the amino acid sequences set forth 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 to 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.

[0098] Mixed Chain Antibodies of the Invention An antibody of the invention may comprise a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 derived from a first antibody in Table 1, 2 or 3, and a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 derived from a second antibody in Table 1, 2 or 3, provided that the first and second antibodies are different. Such antibodies are referred to herein as mixed chain antibodies.

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

[0100] Thus, in one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a first antibody in Table 1, 2 or 3, and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a second antibody in Table 1, 2 or 3, provided that the first and second antibodies are different. The antibody may comprise a heavy chain variable domain amino acid sequence having at least 80% sequence identity to a heavy chain variable domain from a first antibody in Table 1, 2 or 3, and a light chain variable domain amino acid sequence having at least 80% sequence identity to a light chain variable domain from a second antibody in Table 1, 2 or 3, provided that the first and second antibodies are different. 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 a heavy chain variable domain of an antibody in Table 1, 2 or 3 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 a light chain variable domain of an antibody in Table 1, 2 or 3, provided that the first and second antibodies are different.

[0101] The first antibody can be in Table 3 and the second antibody can be in Table 3.

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

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

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

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

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

[0107] 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 described in Table 4. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 4.

[0108] Antibodies derived from IGHV3-53 can be used to generate mixed chain antibodies with antibodies derived 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.” Cell 184(8)(2021):2201-2211). 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). Thus, 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 set forth in Table 5.Thus, an antibody 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 comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 5.

[0109] 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 IGHV 1-58. The resulting mixed chain antibodies are described in Table 6. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 6.

[0110] 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 described in Table 7. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 7.

[0111] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: beta22, beta29, antibody 159 and Omi09. The heavy chain variable domains of these antibodies are derived from IGHV 3-30. The resulting mixed chain antibodies are described in Table 8. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy chain variable domains and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 8.

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

[0113] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: antibody 278, beta44, Omi26, Omi41. The heavy chain variable domains of these antibodies are derived from IGHV 1-18. The resulting mixed chain antibodies are described in Table 10. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 10.

[0114] 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 described in Table 11. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 11.

[0115] In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: Beta56 and Omi23. The heavy chain variable domains of these antibodies are derived from IGHV 4-31. The resulting mixed chain antibodies are described in Table 12. Thus, an antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3) and / or heavy and light chain variable domains, each comprising or consisting of an amino acid sequence having at least 80% (e.g., ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%) sequence identity to the corresponding variable domain of any one of the mixed chain antibodies described in Table 12.

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

[0117] Specific Antibodies of the Invention The invention also provides an antibody that 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, an antibody of the invention comprises a heavy chain variable domain and a light chain variable domain, respectively, of any one of the antibodies in Tables 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and an IgG (e.g., IgG1) constant region.

[0118] For example, the antibody of the present invention can be a full-length Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 or Omi42 antibody. The antibody of the present invention can be a full-length Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi23, Omi28, Omi08, Omi17, Omi29, Omi36 or Omi38 antibody. These antibodies are all highly potent neutralizing mAbs that have been shown to neutralize the Omicron variant of SARS-CoV-2 with an IC50 of 0.1 μg / ml or less. The antibody also has the ability to neutralize, among others, at least the Victoria, alpha, beta, gamma and delta strains of SARS-CoV-2 with an IC50 of 0.1 μg / ml or less.

[0119] The antibody may be derived from the germline heavy chain IGHV1-58 and comprises a proline at position 53 of 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 of 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 Omicron strains (see, e.g., Example 5).

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

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

[0122] 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 the germline heavy chain IGHV1-58, provided that the amino acid at position 58 according to the IMGT numbering is a proline or is substituted with a proline.

[0123] The antibody derived from the 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 Omi-12, beta-47, beta-25, antibody 55, antibody 165, antibody 253, or antibody 318 is described herein (see, e.g., Tables 1-3). The amino acid sequence of the heavy chain variable domain of antibody AZD8895 is provided in SEQ ID NO:963.

[0124] The IGHV1-58 germline V gene sequence encodes the following amino acid sequence: MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAA (SEQ ID NO: 961). Thus, the present invention also provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, 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, with the proviso that the amino acid at position 58 according to the IMGT numbering is a proline or is substituted with a proline.

[0125] 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 NOs: 731, 591, 461, 62, 182, 262, 332 or 963, provided that the amino acid at position 58 according to the IMGT numbering is a proline or is substituted with a proline. The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having SEQ ID NOs: 591, 461, 62, 182, 262 or 332, where the valine at position 58 according to the IMGT numbering is substituted with a proline.

[0126] The antibody may comprise a heavy chain variable domain comprising the amino acid sequence having SEQ ID NO: 963, wherein the isoleucine at position 58 according to the IMGT numbering is substituted with a proline.

[0127] In some embodiments, the antibody derived from the 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 the light chain variable domain of a germline IGLV kappa 3-20 antibody. The germline IGLV kappa 3-20V sequence may encode the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO: 967). Thus, an antibody derived from the 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.

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

[0129] 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, including <10, <9, <8, <7, <6, <5, <4, <3, <2 or 1 modification, provided that the amino acid at position 58 according to the IMGT numbering is a proline or is substituted with a proline. Modified versions of SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963 may include modifications, such as substitutions, deletions and / or additions, described herein.

[0130] The antibody can include an IgG (eg, IgG1) constant region.

[0131] The invention also provides a method for preparing such an antibody. For example, the method may comprise modifying an antibody derived from germline heavy chain IGHV1-58, capable of binding to the spike protein of coronavirus SARS-CoV-2, by substituting the amino acid at position 58 of the heavy chain variable region (according to IMGT numbering) with a proline. 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 sequence of the heavy chain variable domain of each of these antibodies is described herein (see, for example, Tables 1-3 and SEQ ID NO: 963). The invention also provides an antibody obtained or obtained by this method.

[0132] Properties of the Antibodies of the Invention The antibodies of the present invention may be or may include modifications from the amino acid sequence of an antibody in Tables 1-12 while maintaining the activity and / or function of the antibody. Modifications may be substitutions, deletions and / or additions. For example, modifications may include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the amino acid sequence of an antibody in Tables 1-12. For example, modifications may include an amino acid being replaced by an alternative amino acid having similar properties. Some properties of the 20 main amino acids that can be used to select appropriate replacements are as follows:

[0133] [Table 1]

[0134] Modifications can include derivatized amino acids, such as labeled or non-naturally occurring amino acids, provided that the function of the antibody is not significantly adversely affected.

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

[0136] The antibodies of the invention may be modified (e.g., as described above) to improve the efficacy of the antibody or to adapt the antibody to new SARS-CoV-2 variants. Modifications may be amino acid substitutions that adapt the antibody to substitutions in the viral variant. For example, the known binding mode of the antibody to the spike protein (e.g., by crystal structure determination or modeling) may be used to identify amino acids of the antibody that interact with substitutions in the viral variant. This information may then be used to identify possible substitutions in the antibody that will compensate for changes in epitope characteristics. For example, a substitution of a hydrophobic amino acid in the spike protein with an amino acid that changes negatively may be compensated by replacing an amino acid from the antibody that interacts with said amino acid in the spike protein with a positively charged amino acid. The present disclosure encompasses methods of identifying residues of the antibody that may be substituted, for example, by determining the structure of an antibody-antigen complex as described herein.

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

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

[0139] The term "antibody" as used herein can refer to whole antibodies (i.e., comprising two heavy and two light chain elements interconnected by disulfide bonds) and antigen-binding fragments thereof. Antibodies typically comprise immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds to (immunoreacts with) an antigen. "Specifically binds to" or "immunoreacts with" means 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 the binding domains that interact with the antigen. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR).

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

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

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

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

[0144] The antibody may be selected from the group consisting of single chain antibodies, single chain variable fragments (scFv), variable fragments (Fv), fragment antigen binding regions (Fab), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigen binding domain of a natural antibody or an aptamer, single domain antibodies (sdAb), also known as VHH antibodies, nanobodies (single domain antibodies derived from camelids), single domain antibody fragments derived from shark IgNAR, termed VNAR, diabodies, triabodies, anticalins, aptamers (DNA or RNA) and active portions or fragments thereof.

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

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

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

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

[0149] The antibodies of the invention may be full-length antibodies.

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

[0151] An antigen-binding fragment does not necessarily have the same sequence as the parent antibody. In one embodiment, an antigen-binding fragment may have ≧70%, ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, 100% sequence identity with each CDR of the parent antibody. In one embodiment, an antigen-binding fragment may have ≧70%, ≧80%, ≧90%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, 100% sequence identity with each variable region domain of the parent antibody. Typically, the non-identical amino acids of the variable regions are not within the CDRs.

[0152] Antigen-binding fragments of the antibodies of the present invention retain the ability to selectively bind to an 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), and scFv.

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

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

[0155] In terms of functionality, the antibodies of the invention are capable of neutralizing at least one biological activity of SAR-CoV-2 (neutralizing antibodies), in particular neutralizing viral infectivity.

[0156] Neutralization can also be determined using IC50 or IC90 values. For example, the antibody has 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 antibody of the present invention can have an IC50 value of 0.0001 μg / ml to 0.1 μg / ml, sometimes 0.0001 μg / ml to 0.05 μg / ml, or 0.0001 μg / ml to 0.001 μg / ml.

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

[0158] The ability of the antibody to neutralize virus infectivity can be measured using a suitable assay, in particular a cell-based neutralization assay, as shown in the examples. For example, the neutralization ability can be measured by a focus reduction neutralization assay (FRNT), in which the reduction in the number of cells (e.g., human cells) infected with the virus in the presence of the antibody (e.g., at 37°C for 2 hours) is compared to a negative control in which no antibody is added.

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

[0160] Blocking of spike-ACE2 interaction may be total or partial. For example, the antibodies of the invention may reduce spike-ACE2 formation by >50%, >60%, >70%, >80%, >90%, >95%, >99% or 100%. Blocking of spike-ACE2 formation may be measured by any suitable means known in the art, for example by ELISA.

[0161] Most of the antibodies that showed neutralization also showed blocking of the interaction of the spike protein with ACE2. Moreover, some non-neutralizing antibodies are good ACE2 blockers.

[0162] In terms of binding kinetics, the antibodies of the present 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.

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

[0164] Binding affinity (KD) can be quantified by determining the dissociation constant (Kd) and association constant (Ka) for the antibody and its target. For example, an antibody may have an association constant (Ka) of ≥ 10,000 M -1 s -1 , ≧50000M -1 s -1 , ≧100000M -1 s -1 , ≧200000M -1 s -1 Or ≧500000M -1 s -1 and / or the dissociation constant (Kd) may be ≦0.001s -1 , ≦0.0005s -1 , ≦0.004s -1 , ≦0.003s -1 , ≦0.002s -1 Or ≦0.0001s-1 It could be.

[0165] The antibodies of the invention are preferably capable of providing in vivo protection in animals infected with a coronavirus (e.g., SARS-CoV-2). For example, administration of an antibody of the invention to an animal infected with a coronavirus (e.g., SARS-CoV-2) may result in a survival rate of ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90%, ≧95% or 100%. Survival rates may be determined using routine methods.

[0166] An antibody of the invention may have any combination of one or more of the above properties.

[0167] The antibodies of the invention may bind to the same epitope as any one of the antibodies described herein or may compete for binding to the SARS-CoV-2 spike protein (i.e., particularly for antibodies having the heavy and light chain variable regions described above). 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.

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

[0169] The antibodies of the present invention may be modified in the Fc region to improve their stability. Such modifications are known in the art. The 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 modification of the Fc-region. For example, cysteine ​​residues may be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibodies thus generated may have improved internalization capability and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)).

[0170] Alternatively, an antibody can be engineered which has dual Fc regions and may have enhanced complement lysis and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0171] For example, antibodies of the invention may be modified to enhance the interaction of the Fc domain with FcRn. The Fc domain may be modified to improve the stability of the antibody by affecting Fc and FcRn interactions at low pH, for example, in endosomes. M252Y / S254T / T256E (YTE) mutations may be used to improve the half-life of IgG1 antibodies.

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

[0173] In one embodiment, an antibody of the invention comprises an altered Fc domain as described herein below. In another preferred embodiment, an 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.

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

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

[0176] In one embodiment, the antibodies of the invention do not bind to 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 to FcγR but do bind complement.

[0177] In one embodiment, the antibodies of the present invention may generally include modifications that modify the serum half-life of the antibody. Thus, in another embodiment, the antibodies of the present invention have Fc region modifications that modify the half-life of the antibody. Such modifications as well as modifications that modify Fc function may be present. In a preferred embodiment, the antibodies of the present invention have modifications that modify the serum half-life of the antibody.

[0178] In one embodiment, the antibody of the invention may comprise a human constant region, such as an IgA, IgD, IgE, IgG or IgM domain. In particular, human IgG constant region domains, especially IgG1 and IgG3 isotypes, may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.

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

[0180] 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 (C1q), 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 bind to one or more types of Fc receptors.

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

[0182] Some antibodies that selectively bind to FcRn at pH 6.0 but not at pH 7.4 show longer half-lives in various animal models. Some mutations such as T250Q / M428L (Hinton PR.et al.,2004.Engineering human IgG antibodies with longer serum half-lives in primates.J Biol Chem.279(8):6213-6) and M252Y / S254T / T256E+H433K / N434F (Vaccaro C.et al.,2005.Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.Nat Biotechnol.23(10):1283-8), located at the interface between the CH2 and CH3 domains, have been shown to increase the binding affinity to FcRn and extend the half-life of IgG1 in vivo. Thus, there may be modifications at M252 / S254 / T256+H44 / N434 that alter serum half-life, and in particular there may be M252Y / S254T / T256E+H433K / N434F. In one embodiment, it is desired to extend the half-life. In another embodiment, it may actually be desirable to shorten the serum half-life of the antibody, and so modifications that shorten serum half-life may be present.

[0183] A 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.Immunol.166(4):2571-5). Visibly, an alanine substitution at position 333 was reported to increase both ADCC and CDC. Therefore, in one embodiment, there may be modifications at position 333, particularly those that alter the ability to recruit complement. Lazar et al. described a triple mutant (S239D / I332E / A330L) with higher affinity for FcγRIIIa and lower affinity for FcγRIIb, resulting in enhanced ADCC (Lazar GA.et al.,2006). Therefore, there may be modifications of S239 / I332 / A330, especially those that modify the affinity for Fc receptors, especially S239D / I332E / A330L (Engineered antibody Fc variants with enhanced effector function. PNAS 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.,6:3009-3018). Richards et al. investigated a slightly different triple mutant (S239D / I332E / G236A) that improved FcγRIIIa affinity and FcγRIIa / FcγRIIb ratio that mediated enhanced phagocytosis of target cells by macrophages (Richards JO et al 2008.Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells.Mol Cancer Ther.7(8):2517-27).Thus, in one embodiment, the S239D / I332E / G236A modifications may be present.

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

[0185] Major Public V Areas Public V regions (also referred to herein as public V genes) are the V regions of the germline heavy and light chain regions that are found in the majority of antibody responses to SARS-CoV-2 found in a population. In this application, the V regions are specific responses to beta SARS-CoV-2 variants. That is, many individuals utilize the same v regions from their germline v region repertoire when generating an immune response to SARS-CoV-2 variants.

[0186] As used herein, an antibody "derived from" a particular v-region refers to an antibody generated by V(D)J recombination with that germline v-region sequence. For example, a germline IGHV3-53 v-region sequence may undergo somatic recombination and somatic mutation to arrive at an antibody that specifically binds to the spike protein of SARS-CoV-2. Although the nucleotide sequence encoding the antibody is unlikely to contain an identical sequence to the IGHV3-53 germline sequence, the antibody is nevertheless still derived from this v-region. An antibody of the invention typically contains no more than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 non-silent mutations in the v-region when compared to the germline sequence. An antibody of the invention typically contains no more than 2-20 non-silent mutations in the v-region when compared to the germline sequence, such as no more than 5-15, 6-13, 7-12 non-silent mutations. Germline v-region sequences are well known in the art and methods for identifying whether a particular region of an antibody is derived from a particular germline v-region sequence are also well known in the art.

[0187] 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 have found that the potent neutralizing antibodies identified herein contain relatively few mutations within the CDRs of these v-regions. Thus, in one embodiment, the antibody of the invention is encoded by a v-region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV1-69, IGHV3-30, IGHV3-33, IGHV1-18, IGHV13-9 or IGHV4-31 and has 2-20 non-silent nucleotide mutations or 5-15 non-silent mutations, 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, when compared to a naturally occurring germline sequence. A silent mutation, as defined herein, is a change in a nucleotide sequence that does not result in a change in the amino acid sequence encoded by the nucleotide sequence. Thus, a non-silent mutation is a mutation that causes a change in the amino acid sequence encoded by the nucleotide sequence.

[0188] The inventors have surprisingly found that the light chain variable regions of two antibodies with the same heavy chain v region can be exchanged to generate a mixed chain antibody comprising the heavy chain variable region of a first antibody and the light chain variable region of a second antibody. For example, the two antibodies can both comprise heavy chain variable regions derived from IGHV3-53. Preferably, both antibodies also comprise light chain variable regions derived from the same light chain v region, but this is not essential, since for example the light chain of antibody 222 can be matched with any heavy chain variable region derived from IGHV3-53, leading to a potent neutralizing antibody. As described above, the two antibodies can comprise heavy chain variable regions derived from IGHV3-53 and / or IGHV3-66.

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

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

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

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

[0193] Antibody conjugates The present invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof) or an immunoconjugate comprising a radioisotope (i.e., a radioconjugate). Conjugates of antibodies and cytotoxic agents can be formed using a variety of bifunctional protein coupling agents known in the art.

[0194] The antibody of the present invention may be conjugated to a molecule that modulates or alters serum half-life. The antibody of the present invention may bind to albumin, for example, to modulate serum half-life. In one embodiment, the antibody of the present invention will also comprise a binding region specific for albumin. In another embodiment, the antibody of the present invention may comprise a peptide linker that is an albumin-binding peptide. Examples of albumin-binding peptides are listed in WO2015 / 197772 and WO2007 / 106120, which are incorporated by reference in their entirety.

[0195] Polynucleotides, Vectors and Host Cells The present invention also provides one or more isolated polynucleotides (e.g., DNA) encoding an antibody of the invention. In one embodiment, the polynucleotide sequences are present together on multiple polynucleotides, but together can encode an antibody of the invention. For example, a polynucleotide can encode the heavy chain variable region and / or the light chain variable region of an antibody of the invention. A polynucleotide can encode the full-length heavy chain and / or light chain of an antibody of the invention. Typically, one polynucleotide encodes each of the heavy and light chains.

[0196] 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 antibody heavy and light chains can be synthesized from the corresponding amino acid sequences, if necessary. The general methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to "Current Protocols in Molecular Biology", 1999, FMAusubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing. The polynucleotides of the present invention can be provided in the form of an expression cassette, which comprises a regulatory sequence operably linked to the inserted sequence, thereby allowing the in vivo expression of the antibodies of the present invention. 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 typically, in turn, provided within a vector (e.g., a plasmid or a recombinant viral vector). Thus, in one embodiment, the present invention provides a vector encoding the antibodies of the present invention. In another embodiment, the present invention provides a vector that collectively encodes the antibodies of the present invention. The vector can be a cloning vector or an expression vector. Suitable vectors may be any vector that can carry a sufficient amount of genetic information and allow expression of the polypeptide of the present invention. The polynucleotide, expression cassette or vector of the present invention is introduced into a host cell, for example, by transfection. Thus, the present invention also provides a host cell comprising one or more polynucleotides, expression cassettes or vectors of the present invention. The polynucleotide, expression cassette or vector of the present invention may be introduced into a host cell transiently or permanently to allow expression of the antibody 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.Particular examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NS0 and COS cells or any other cell line used herein, such as those used in the Examples. Preferably, the cell line selected is one that allows not only stable but also mature glycosylation.

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

[0198] Antibody Combinations The inventors have found that certain antibodies of Table 3 and certain combinations of antibodies of Tables 3, 2 and 1 are particularly effective when used in combination, 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 that do not bind non-overlapping epitopes.

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

[0200] The antibody combinations of the invention may be useful as therapeutic cocktails. As such, the invention also provides pharmaceutical compositions comprising the antibody combinations of the invention, as further described below.

[0201] The antibody combination of the present invention may be useful for diagnosis. Therefore, the present invention also provides a diagnostic kit comprising the antibody combination of the present invention. Provided herein is also a method for diagnosing a disease or complication associated with coronavirus infection in a subject, as further described below. A fully cross-neutralizing antibody, such as Omi03, may be used as a reference to confirm the presence and / or amount of variant of concern (VoC) SARS-CoV-2 in a sample. To confirm the presence and / or amount of VoC in a sample, an antibody that binds to a limited number of VoCs may be used. For example, if Omi03 shows binding to the sample but Omi24 does not show binding to the sample of SARS-CoV-2, the spike protein may be a spike protein of delta VoC. This may be determined by any method known to the skilled artisan, for example via an immunoassay, such as an ELISA or immunochromatographic assay. The reduction in binding may be determined by comparison and / or normalization to a reference and / or comparison to positive / negative control samples or data.

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

[0203] The compositions herein may contain 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 undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0204] Suitable pharma- ceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water and saline.

[0205] Other suitable pharma- ceutically acceptable carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) 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, for example, sugars, polyalcohols, for example, mannitol, sorbitol, or sodium chloride in the composition.

[0206] Pharmaceutical compositions 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 to high drug concentration.

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

[0208] The additional therapeutic agent can be an anti-inflammatory agent, such as a corticosteroid (e.g., dexamethasone) or a non-steroidal anti-inflammatory drug (e.g., tocilizumab).

[0209] 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 present invention is a kit comprising the antibody or other composition of the present invention and instructions for use. The kit may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents discussed herein.

[0210] Methods and uses of the present invention The present invention further relates to the use of the antibodies, antibody combinations and pharmaceutical compositions described herein in methods of treatment of the human or animal body, for example by therapy or in diagnostic methods. Methods of treatment can be therapeutic or prophylactic.

[0211] For example, the invention relates to a method of treating a coronavirus (e.g., SARS-CoV-2) infection or a disease or complication associated therewith, such as COVID-19. The method may comprise administering a therapeutically effective amount of an antibody, antibody combination or pharmaceutical composition of the invention. The method may further comprise identifying the presence of a coronavirus, such as SARS-CoV-2 or a fragment thereof, in a sample from the subject. The invention also relates to an antibody, antibody combination or pharmaceutical composition according to the invention for use in a method of treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith, such as COVID-19.

[0212] The present invention also relates to a method for formulating a composition for treating a coronavirus (e.g., SARS-CoV-2) infection, a disease or complication associated therewith, such as COVID-19, said method comprising mixing an antibody, antibody combination or pharmaceutical composition according to the invention with an acceptable carrier to prepare said composition.

[0213] The present invention also relates to the use of an antibody, an antibody combination or a pharmaceutical composition according to the invention for treating a coronavirus (e.g., SARS-CoV-2) infection or a disease or complication associated therewith, such as COVID-19.

[0214] The invention also relates to the use of an antibody, an antibody combination or a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment or prevention of a coronavirus (e.g. SARS-CoV-2) infection or a disease or complication associated therewith, such as COVID-19.

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

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

[0217] The SARS-CoV-2 strain can include, for example, one or more mutations in the spike protein compared to hCoV-19 / Wuhan / WIV04 / 2019 (WIV04) (GISAID accession no. EPI_ISL_402124). In other words, the SARS-CoV-2 strain can be, for example, a modified hCoV-19 / Wuhan / WIV04 / 2019 (WIV04) strain that includes one or more modifications in the spike protein.

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

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

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

[0221] The methods and uses of the invention may include improving or reducing the severity, duration or frequency of symptoms of a disease state (e.g., COVID-19) (e.g., less pain or discomfort), which may or may not result in a direct impact on the disease. Symptoms or complications may be fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, respiratory tract infection, cytokine storm, acute respiratory distress syndrome (ARDS), sepsis and / or organ failure (e.g., cardiac, renal, hepatic, GI, pulmonary).

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

[0223] The methods and uses of the invention may include preventing a coronavirus infection from occurring in a subject (e.g., a human), particularly if such a subject is predisposed to complications associated with coronavirus infection.

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

[0225] The present invention relates to 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, 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 a non-silent mutation that may affect the efficacy of existing treatments for SARS-CoV-2 infection. Typically, the non-silent mutation is on an epitope recognized by prior art antibodies that specifically bind to an epitope of SARS-CoV-2 and / or the antibodies described herein, for example on the spike protein of SARS-CoV-2. If the antibody does not bind to the target, this may indicate that the target contains a mutation that may alter the efficacy of an existing SARS-CoV-2 treatment.

[0226] 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, the presence of which indicates that the subject is infected with SARS-CoV-2.

[0227] 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 blot, immunoprecipitation and immunofluorescence. In vivo techniques involve introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location within the subject can be detected by standard imaging techniques. Detection techniques can provide a qualitative or quantitative readout, depending on the assay used.

[0228] Typically, the present invention relates to methods and uses for human subjects in need thereof. However, non-human animals, such as rats, rabbits, sheep, pigs, cows, cats or dogs, are also contemplated. Subjects, such as health care workers or people who have been in contact with infected individuals, may be at risk of exposure to coronavirus infection. Subjects may have visited or be planning to visit countries where coronavirus outbreaks are known or suspected. Subjects may also be at higher risk, such as immunocompromised individuals, such as those undergoing immunosuppressive therapy or those suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS). Subjects may be asymptomatic or pre-symptomatic.

[0229] The subject may be in the early, middle or late phase of the disease.

[0230] The subject may be hospitalized or in the community at the time of initial onset and / or may be hospitalized subsequently.

[0231] The subject may be male or female.

[0232] In certain embodiments, the subject is typically male. The subject may not be infected with coronavirus, such as SARS-CoV-2. The subject may have a predisposition to more severe symptoms or complications associated with coronavirus infection. The method or use of the present invention may include a step of identifying whether the patient is at risk of developing more severe symptoms or complications associated with coronavirus.

[0233] In embodiments of the invention relating to prevention or treatment, the subject may or may not have been diagnosed with a coronavirus, e.g., SARS-CoV-2, infection.

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

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

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

[0237] The dose of the antibody may vary depending on the age and size of the subject, as well as the disease, the condition and route of administration. The antibody may be administered at a dose of about 0.1 mg / kg body weight to about 100 mg / kg body weight, for example, at a dose of about 5 mg / kg to about 10 mg / kg. The antibody may also be administered at a dose of about 50 mg / kg body weight, 10 mg / kg, or about 5 mg / kg.

[0238] The combinations of the invention can be administered, for example, at a dose of about 5 mg / kg to about 10 mg / kg per antibody, or at a dose of about 10 mg / kg or about 5 mg / kg per antibody. Alternatively, the combinations can be administered at a total dose of about 5 mg / kg (e.g., a dose of 1.67 mg / kg of each antibody for a three antibody combination).

[0239] The antibody or antibody combination of the invention may be administered in a multiple dose regimen. For example, an initial dose may be followed by administration of a second or multiple subsequent doses. The second or subsequent doses may be separated by an appropriate amount of time.

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

[0241] Coadministration of two or more agents and / or antibodies can be accomplished in a number 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.

[0242] For example, an antibody of the invention can be administered before, after, or concurrently with another antibody or binding fragment thereof of the invention. Particularly useful combinations are, for example, described above.

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

[0244] In embodiments in which the invention relates to detecting the presence of a coronavirus, such as SARS-CoV-2, or a protein or fragment thereof, in a sample, the antibody contains a detectable label. Methods are known in the art for attaching a label to an antibody, for example, by direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody. Alternatively, the antibody can be indirectly labeled, for example, by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody with a fluorescently labeled secondary antibody and end-labeling of a biotinylated DNA probe such that it can be detected by fluorescently labeled streptavidin.

[0245] Detection may further include (i) an agent known to be useful in detecting the presence of a coronavirus, e.g., SARS-CoV-2 or a protein or fragment thereof, such as an antibody against another epitope of the spike protein or another protein of the coronavirus, e.g., an anti-nucleocapsid antibody; and / or (ii) an agent known to be incapable of detecting the presence of a coronavirus, e.g., SARS-CoV-2 or a fragment thereof, i.e., providing a negative control.

[0246] In certain embodiments, the antibody has been modified to increase its stability. Suitable modifications are described above.

[0247] The present invention also encompasses kits 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 combination of labeled antibodies of the present invention; a means for determining the amount of coronavirus, e.g., SARS-CoV-2, in a sample; and a means for comparing the amount of coronavirus, e.g., SARS-CoV-2, in a sample to a standard. The labeled antibody or combination of labeled antibodies may be packaged in a suitable container. The kit may further include instructions for using the kit to detect coronavirus, e.g., SARS-CoV-2, in a sample. The kit may further include other agents known to be useful in detecting the presence of coronavirus, as set forth above.

[0248] For example, the antibody or combination of antibodies of the present invention is used in a lateral flow test. Typically, a lateral flow test kit is a handheld device that includes a series of capillary beds, such as pieces of porous paper, absorbent pads based on microstructured or sintered polymers. The test runs a liquid sample along the surface of the pad with reactive molecules to give a visible positive or negative result. The test may further include using other agents known to be useful for detecting the presence of coronaviruses, such as SARS-CoV-2 or fragments thereof, as indicated above, such as anti-nucleocapsid antibodies.

[0249] others It should be understood that the various uses of the disclosed antibody combinations or pharmaceutical compositions of the present invention can be tailored to the specific needs of the art. It should also be understood that the terminology used herein is only for describing certain embodiments of the present invention and is not intended to be limiting. In addition, the singular forms "a," "an," and "the" used in this specification and the appended claims include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "antibody" includes two or more "antibodies."

[0250] Furthermore, when reference is made herein to "≧x," this means greater than or equal to x. When reference is made herein to "≦x," this means less than or equal to x.

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

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

[0253] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0254] The following examples illustrate the invention. EXAMPLES

[0255] Example 1. Generation of antibodies specific to early pandemic SARS-CoV-2 and beta SARS-CoV-2 strains The antibodies in Table 1 relate to a set of mAbs generated against early pandemic strains of SARS-CoV-2. The antibodies in Table 2 relate to a set of mAbs generated against beta strains of SARS-CoV-2.

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

[0257] Example 2. Generation of antibodies specific to the Omicron strain of SARS-CoV-2 Omicron BA.2 lineage Omicron BA.2 was first reported from South Africa on 17 November 2021, around the same time that Omicron BA.1 was reported. BA.2 has increased relative to BA.1 in several countries, including Denmark, India and the UK, and currently accounts for the majority of Omicron infections in Denmark. There is accumulating evidence that BA.2 is more transmissible than BA.1, but there is no evidence of increased disease severity.

[0258] BA.2 is related to BA.1 and shares 21 amino acid substitutions spread across S, but with some 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 the unique mutations S371L, G446S and G496S, and in some isolates R346K (BA.1.1), while BA.2 contains S371F, T376A, D405N and R408S. All of these residues may have different effects on antibody binding and may modulate neutralization. This is particularly true for BA.1 G446S, G496S, BA.2 D405N, R408S, which are at the edge of the ACE2 binding footprint, and for BA.1.1, the R346K change is close to the N343 glycan and may modulate potent antibody binding to this region. BA.3 does not contain the unique mutations associated with BA.1 and BA.2, but appears to be a fusion of the two, resembling BA.1 at the N-terminus and switching from the mutation G496S to one resembling BA.2 at the C-terminus.

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

[0260] Two additional mutations in the RBD are of most concern from the perspective of antibody escape: L452R is a chemically radical change and is one of a pair of changes in the delta RBD (the other, T478K, has already been found in the Omicron lineage). The mutation F486L was found in sequences of SARS-CoV-2 isolated from mink early in the pandemic and is also a site of escape mutation for some mAbs (Gobeil et al., 2021, “Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity”. Science 373, 6555). The F486V change in BA.4 / 5 is also a reduction in the bulk of the hydrophobic side chain, similar to F486L, but more pronounced. Residues 452 and 486 are both near the edge of the ACE2 interaction surface (Figure 9B) and together with the reversion to the ancestral sequence Q493, both of which are 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 further increase antibody escape, while the reversion at 493 may decrease escape from prior viral responses.

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

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

[0263] Using neutralization assays, it was shown that Delta infection alone does not provide protection (neutralization) against BA.2.75. Mutations in BA.2.75 result in lower neutralization titers of vaccine sera compared to BA.2. Although individual mutations in BA.2.75 can cause a large reduction in neutralization titers compared to the full BA.2.75S sequence, these are balanced by the R393Q reversion mutation, which may have been selected to increase affinity for ACE2 and enhance transmissibility of BA.2.75. Further evolution of the Omicron lineage is inevitable and there are many trade-offs between antibody escape and ACE2 affinity that seem likely to result in successive waves of infection.

[0264] Emerged sublineages BA.2, BA.4, and BA.5 Several lineages have grown rapidly from within both the BA.2 and BA.5 branches. Most notable is the large degree of convergent evolution, particularly at antigenic RBD positions such as 346, 444, 452, 460, 486, 490, 493 and 494. These lineages include the BA.4 / 5 branch (naturally containing L452R, F486V and the revertant R493Q), e.g. BA.4.6 and BF.7 (R346T), BA.4.7 (R346S), BQ.1 (K444T, N460K) and BQ.1.1 (R346T, K444T, N460K); the BA.2.75 branch (G339H, G446S, N460K and the revertant R493Q); Examples include those from BA.2.75.2 (containing the R346T and F486S and BA.2.75 mutations), BN.1 (also known as BA.2.75.5.1 and containing the R346T, K356T, F490S and BA.2.75 mutations), and BM.1.1.1 (also known as BA.2.75.3.1.1.1 and containing the R346T, F486S, F490S and BA.2.75 mutations). Examples of several other second generation BA.2 variant strains include 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), BS.1 (also known as BA.2.3.2.1; R346T, L452R, N460K, G476S, and There are also BJ.1 x BM.1.1.1 (also known as BA.2.75.3.1.1.1) recombinants, XBB (containing R346T, L368I, V445P, G446S, N460K, F486S, F490S and Q493 reversions relative to BA.2), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and Q493R reversions) and finally

[0265] Outside the RBD, convergent evolution is less frequent but still present. Many second generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in 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).

[0266] Potent neutralizing antibodies isolated after infection with Omicron. Five volunteers who had recovered from sequence-confirmed Omicron infection were recruited, and samples were collected 10–14 days after symptom onset. All volunteers had received two 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 one amino acid substitution in the S NTD (S247R) compared to the sequence of the Wuhan strain used in all current vaccines. In all cases, focus reduction neutralization 50% titers (FRNT50) against Omicron were above 100, although at this early time point, titers were significantly lower than those against Victoria (Figure 1A).

[0267] B cells from five donors were stained with full-length BA.1 trimer and single cells were sorted by FACS (Figure 1B). After degenerate RT-PCR reactions, 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 against full-length BA.1 or wild-type S (WT Wuhan) along with the BA.1 RBD and NTD. A total of 1,122 single cells were sorted and 545 mAbs were recovered.

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

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

[0270] A survey of gene usage (Figure 1D, Table 17) revealed that 9 / 28 mAbs belonged to the VH3-53 and related VH3-66 gene families. VH3-53 and VH3-66 have been repeatedly isolated in SARS-CoV-2 infections, where they form public antibody responses and play a role in binding to the neck site of the RBD to block ACE2 binding. In the past, it was observed that many VH3-53 and VH3-66 mAbs lost activity against VoCs containing the N501Y mutation, while some VH3-53 antibodies (mAb222 and beta-27) were completely resistant to the N501Y change found in alpha, beta, and gamma, but their activity against omicron BA.1 or BA.2 was knocked down.

[0271] Approximately half of the gene families observed in the potent pandemic early antibodies (Table 1) are also represented in the Omicron set (Figure 1C), with perhaps the most striking difference being that VH1-69 is absent from the early antibodies but is present in 6 / 28 (2, 24, 30, 31, 34 and 38) of the potent Omicron set, and we also found this in two beta antibodies, beta49 and 50, that bind to a site proximal to the N343 glycan. Analysis of Omicron mAbs showed that, for beta49, 50, the CDR3 sequence is much longer, suggesting a different binding mode. In the beta set of mAbs, the expansion of the public response was found to be mediated through VH4-39 (6 / 27 mAbs), which binds to an epitope surrounding the 501Y mutation, although the majority lose activity against BA.1. It is noteworthy that none of the current set of Omicron mAbs is encoded by VH4-39.

[0272] Compared to the early pandemic antibody set, we found higher levels of somatic mutations in both the heavy and light chains of mAb Omicron compared to the early pandemic set (means of VH and VL 9.00 and 6.00, respectively, compared to early pandemic 4.55 and 4.25). These results would be consistent with the evolution of increased Omicron affinity through somatic mutations in vaccine-induced memory B cells.

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

[0274] The data provided in Tables 13, 14 and 16 includes some IC50 data obtained using pseudoviral constructs. The data in Table 18 consists of IC50 results obtained only from the true viral constructs.

[0275] 17 / 28 antibodies are cross-reactive against all VoCs, with less than 10-fold difference in FRNT50 titers between all viruses. Omi-06, 24, 30, 31, 34 and 41 have reduced or no activity against delta, and 3 / 6 of these belong to the VH1-69 family and may have epitopes that affect the L452R delta mutation (delta and BA.1 share T478K). Antibodies Omi-09 and 32 perform poorly against beta and gamma, and may be sensitive to the E484K found in beta and gamma, but may tolerate the E484A change in omicron (omicron shares N501Y and K417N with beta, and gamma is N501Y, K417T). Finally, 129 anti-NTD mAbs were isolated, but only one of these, Omi-41, showed a FRNT50 titer of less than 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 mutations found in delta.

[0276] Neutralization of BA.1 compared to BA.1.1, BA.2, and BA.3 Lentivirus-based reporters were constructed pseudotyped with the S gene sequences of Victoria, BA.1, BA.1.1, BA.2 and BA.3. Neutralization assays against Omicron mAbs are shown in Figure 2B and Tables 14 and 18, and most antibodies showed little difference in neutralization of BA.1, BA.1.1, BA.2 and BA.3. However, there were some notable exceptions. Neutralization of BA.2 was reduced 38, 3 and 158 fold compared to BA.1 with Omi-8, 29 and 32, respectively, whereas neutralization of BA.1.1 was reduced 40.9, 10.8, 7.8 and 6.6 fold compared to BA.1 with Omi-6, 24, 34 and 35, respectively, and knocked out with Omi-39 and 40. Neutralization of BA.3 by Omi-mAbs was similar to that seen with BA.2, with the exception of Omi-06 and Omi-36, which had significantly lower BA.3 neutralization titers than BA.1 or BA.2. For some reason, the NTD-binding mAb Omi-41 did not neutralize Victoria in the pseudovirus system, but did neutralize live virus. This was also found with the early pandemic mAb 159, which showed strong activity against live virus but not pseudovirus.

[0277] Pseudovirus neutralization curves for a panel of mAbs isolated from early pandemic cases and for mAbs isolated from beta cases are shown in Figure 4A,B and Table 15. In most cases, neutralization titers against BA.1, BA.1.1 and BA.2 are similar, but there are some differences, with mAbs 40, 278 and 318 neutralizing BA.2 better than BA.1, whereas 222, beta22, 29, 54, 55 and 56 neutralizing BA.1 better than BA.2, and beta53, which binds close to the N343 glycan, showing reduced neutralization of BA.1.1.

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

[0279] In summary, the neutralization of most Omicron monoclonal antibodies is not affected by the differences between the BA.1, BA.1.1, BA.2, or BA.3 mutations. However, some monoclonal antibodies, particularly known antibody A (REGN10987) and known antibody C (AZD1061), which neutralize BA.2 more readily than BA.1, and known antibody K (S309), which shows reduced neutralization of BA.2, show differences and subline typing may be encouraged prior to use. A structural explanation for the differences between BA.1, BA.1.1, BA.2, and BA.3 neutralization is described below.

[0280] Neutralization of BA.1, BA.1.1, BA.2, and BA3 by immune sera To determine whether the difference in transmissibility of BA.1 and BA.2 could be due to differences in neutralization and also whether BA.2 could escape the BA.1 antibody response, we performed neutralization assays using sera from various sources. First, we performed neutralization assays for Victoria, BA.1, BA.1.1, BA.2 and BA.3 using sera collected from vaccinees who received the Oxford / AstraZeneca AZD1222 (n=41) or Pfizer / BioNtech BNT162b2 (n=20) vaccines (Figure 3A,B).

[0281] For AZD1222, samples were taken 4 weeks after the second and third doses of the vaccine. After the third dose of AZD1222, there was a small but significant difference between pseudovirus neutralization with reduced titers against BA.2 vs. BA.1 (1.17-fold, p=0.0019) and BA.1.1 vs. BA.1 (1.29-fold, p=0.0086). For BNT162b2, samples were taken 4 weeks and 6 months after the second dose of the vaccine, before the third dose, and 4 weeks after the third dose. After the third dose of the vaccine, titers against BA.1, BA.2, and BA.1.1 were similar with no significant differences between them. Next, the neutralization profile of sera taken from cases infected with Omicron was determined. Early samples (n=12) were collected <14 days (median 13 days) from symptom onset, whereas late samples (n=17) were collected >21 days (median 38 days) from symptom onset. All cases had received at least two doses of vaccine, with some recovered late cases receiving a third dose of vaccine after infection with Omicron. Neutralization against Victoria, alpha, beta, gamma, delta, and Omicron was tested using a live virus neutralization assay (Figure 3C). At early time points, all vaccinated cases had high titers against Victoria, with geometric mean FRNT50s approaching 1 / 3000, and demonstrated broad neutralization of VoC with FRNT50s >1 / 1000 for all viruses except Omicron (FRNT50=558). At later time points, titers against Victoria did not change, but titers against VoC and Omicron increased (3-fold, p=0.0123). Pairwise comparison of early and late samples from the same individuals confirmed widespread enhanced responses following Omicron infection (Fig. 5A ).

[0282] Neutralization of Victoria, BA.1, BA.1.1, BA.2, and BA.3 was assayed by pseudovirus neutralization. BA.1 neutralization titers were higher at later time points. However, sera were all obtained from BA.1-infected cases, and BA.2 vs. BA.1 neutralization titers were slightly but significantly decreased (1.7- and 1.5-fold at <14 and >21 days, respectively, p=0.0034 and 0.0067), BA.1.1 vs. BA.1 titers were not significantly decreased, but BA.3 vs. BA.1 titers were decreased 1.7-fold at >21 days (p=0.0012) (Figures 3D, 5B).

[0283] In summary, after three doses of the vaccine, especially BNT162b2, good neutralizing titers of antibodies against Omicron BA.1, BA.1.1, BA.2 and BA.3 were induced, with only minor differences between titers against BA.1, BA.1.1, BA.2 and BA.3. This may indicate that the increased transmissibility of BA.2 is not due to increased vaccine escape. After the breakthrough of Omicron infection, previously vaccinated individuals will have augmented broad antibody responses against variants of concern and a robust response against Omicron. As there are only minor differences in neutralization of BA.1 and BA.2, it seems unlikely that BA.2 superinfection will occur in BA.1-exposed and vaccinated cases, at least in the short term.

[0284] Neutralization of BA.4 compared to BA.1, BA.1.1, BA.2 and BA.3 Neutralization of BA.4 / 5 was evaluated in comparison to Omicron sublineages BA.1, BA.1.1, BA.2, BA.3, and early pandemic Victoria strains. BA.4 / 5 was shown to have a more extreme antibody escape phenotype than BA.1 and BA.2, with sera from three-vaccinated donors showing approximately 2- to 3-fold reduced neutralization titers compared to BA.1 and BA.2 neutralization. Furthermore, sera from breakthrough BA.1 infections in vaccinees showed approximately 2- to 3-fold 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 at preventing transmission of BA.4 / 5. Thus, new monoclonals and combinations that fill this gap may be needed to protect extremely vulnerable individuals and those unable to mount an adequate vaccine response.

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

[0286] Neutralization assays were performed using sera obtained 28 days after the third dose of the Oxford-AstraZeneca vaccine ADZ1222 (n=41) (Flaxman et al.,2021. “Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002)”. Lancet 398,981-990.) or the Pfizer-BioNtech vaccine BNT162b2 (Cele et al.,2021; “Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization”. Nature 602,654-666) (n=20) (Figure 7A,B). For AZD1222, neutralization titers for BA.4 were reduced 2.1-fold compared to BA.1 (p=0.0001) and 1.8-fold compared to BA.2 (p=0.0001). For BNT162b2, neutralization titers were reduced 3.2-fold (p=0.0001) and 3.1-fold (p=0.0001) compared to BA.1 and BA.2, respectively. These declines in titers may reduce vaccine efficacy, especially in the long term, as antibody titers naturally decline.

[0287] Neutralization of BA.4 / 5 by sera from breakthrough BA.1 infection Vaccinated volunteers with breakthrough Omicron infection were recruited at the start of the Omicron outbreak. Samples were first collected within 14 days of symptom onset (median 13 days) and late samples were collected 21 days or more after symptom onset (median 38 days) n=16. Pseudovirus neutralization assays were performed against a panel of pseudoviruses representing variants of concern and Omicron sublineages (Figure 7C,D).

[0288] BA.1 infection after vaccination resulted in broad neutralizing responses with high titers against all VoC, which were 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 BA.1 and BA.2, with BA.4 / 5 titers reduced 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. At later time points, BA.4 / 5 titers were reduced 3.4-fold (p=0.0001) and 2-fold (p=0.0017) compared to BA.1 and BA.2, respectively.

[0289] Thus, BA.4 / 5 shows some immune escape from the vaccine / BA.1 response compared to BA.1 and BA.2, and because these samples were all taken reasonably close to the time of infection, further decline over the following months may render the patient susceptible to reinfection with BA.4 / 5.

[0290] BA.4 / 5 escape from monoclonal antibodies Sensitivity to L452R: So far, it has been reported that Omi-24, 30, 31, 34 and 41 show a complete knockout of neutralizing activity against Delta, while Omi-06 shows a severe knockdown of activity (Nutalai et al., 2022). Since BA.1 and BA.2 contain only one of the two Delta RBD mutations (T478K), while BA.4 / 5 also contains L452R, one would expect that all five of these L452-directed mAbs would be knocked out in BA.4 / 5. This has indeed been observed (Figure 8A, Table 20). Omi-41 also fails to neutralize, which may be due to differences in the mutations in the NTD (Figure 9A).

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

[0292] Since detailed information is available on the interaction of several omicron-responsive antibodies with the RBD, the BA.4 / 5 RBD mutations were modeled in relation to the known structures of omicron Fabs 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, 467-484 e415; Nutalai et al., 2022), (Figure 11). The Omi-31 complex is shown in Figure 11A, showing that L452 fits snugly into a hydrophobic pocket and cannot accommodate the larger positively charged arginine in BA.4 / 5 and delta.

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

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

[0295] Activity of commercial antibodies against BA.4 and BA.5 A panel of antibodies developed for therapeutic / prophylactic use was tested against BA.4 / 5 (Figure 8B, Table 21). Many of these antibodies already have significantly 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 (less than 2-fold reduction), while for AZD8895, residual activity against BA.2 was knocked out. The activity of the combination of both 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 (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 against BA.2 in BA.4 / 5, and similarly, the remaining BA.1 neutralizing activity in BA.4 / 5 was knocked out for ADG20 (Yuan et al., 2022, “A broad and potent neutralization epitope in SARS-related coronaviruses”. bioRxiv. https: / / doi.org / 10.1101 / 2022.03.13.484037). Regarding S309 (VIR-7831 / 7832) (Sun and Ho, 2020, “Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic”. Antib Ther 3, 246-256.), activity against BA.4 / 5 was reduced by 1.6-fold compared to BA.2.

[0296] These effects can be rationalized by reference to the way antibodies interact with the RBD; for example, in the case of AZD8895 (an IGHV1-58 genotypic mAb, Figure 11E), F486 forms a hydrophobic interaction hotspot that is abolished by mutation to a 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, 2183-2200 e2122; Liu et al., 2021, “The Beta mAb response underscores the antigenic distance to other SARS-CoV-2 variants”. Cell, Host and Microbe 30, 53-68), which explains the significant impact of the F486V mutation on neutralization of these mAbs (Figure 8A, 13).

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

[0298] Neutralization of BA2.75 by sera from vaccine breakthrough BA.1 or BA.2 infections Breakthrough BA.1 serum samples were collected from vaccinated volunteers 28 days or more after symptom onset (median 38 days, n=16). Pseudovirus neutralization assays were performed against the panel of pseudoviruses described above (Figure 17C). Neutralization titers of BA.2.75 were similar to BA.2 and 1.4-fold (p=0.0052) and 2.0-fold (p=0.0001) higher than BA.2.12.1 and BA.4 / 5, respectively, suggesting that BA.2.75 may be less likely to cause reinfection in individuals with a BA.1 breakthrough infection than BA.2.12.1 or BA.4 / 5.

[0299] Breakthrough BA.2 serum samples were collected from vaccinated volunteers 12 days or more after symptom onset (median 29 days, n=23). Pseudovirus neutralization assays were performed against a panel of pseudoviruses Victoria, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5 and BA.2.75 (Figure 17D). Here, neutralization titers against BA.2.75 were significantly reduced compared to BA.2 (1.4-fold, P=0.0021) and similar to BA.2.12.1, but still higher than BA.4 / 5 (1.4-fold, P=0.0123). Taken together, BA.2.75 shows a degree of escape from the humoral response induced by BA.2 breakthrough infection but not BA.1 infection.

[0300] Individual BA.2.75 mutations have different effects on neutralization To understand the impact of individual mutations in the BA.2.75 RBD, these mutations were introduced individually into the pseudovirus BA.2 background and neutralization was assayed using Pfizer BNT162b2 sera vaccinated three times (Figure 17E). BA.2 neutralization titers were reduced by 3 / 4 BA.2 single mutation variants, the most reduced by N460K (3.1-fold, p<0.0001), followed by D339H (1.3-fold, p=0.0006) and G446S (1.2-fold, p=0.2312), but neutralization titers were increased 1.5-fold by the R493Q reversion mutation (p<0.0001). Q493 is present in all vaccines, explaining why vaccine sera show increased activity against this reversion mutation.

[0301] Escape from monoclonal antibodies in BA.2.75 To analyze how BA.2.75 affects neutralizing antibody activity, a pseudovirus assay was used to test a recently reported panel of potent human mAbs generated from a case of Omicron breakthrough infection (BA.1 IC50 titer <0.1 μg / ml) (Nutalai et al., 2022). (Figure 19A, Table 22). Of the 27 RBD-specific mAbs, those belonging to the IGHV3-53 / 66 family are most severely affected. Three (Omi-16, Omi-29, and Omi-36) showed a complete knockout of BA.2.75 neutralization. Four more (Omi-18, Omi-20, Omi-27 and Omi-28) showed a greater than 5-fold reduction compared to BA.2, consistent with the observation that in the structure of the RBD / IGHV-3 / 66 complex, N460 interacts with a highly conserved GGS / T motif in CDR-H2 (Figure 21B) (Dejnirattisai et al. 2021, Liu et al. 2021, Nutalai et al. 2022).

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

[0303] Omi mAbs were also tested against pseudoviruses encoding single point mutations in the BA.2 RBD mentioned above (Figure 23, Table 24). VH3-53 / 66 mAbs that lost neutralization against BA.2.75 were also affected by the N460K mutation, confirming the prediction that this residue is important for binding of some members of this public gene family. Interestingly, the BA.2+N460K mutation alone shows a greater impact on the activity of some mAbs than BA.2.75: the neutralization titer of Omi-03 (IGHV3-53) was reduced 50-fold with BA.2+N460K but only 2-fold with BA.2.75. Omi-17 (IGHV3-66) was completely knocked out with BA.2+N460K but only 4-fold with BA.2.75. Omi-33 (IGHV3-33) was reduced 7-fold in BA.2+N460K but was unchanged in BA.2.75, so other mutations in BA.2.75, particularly the R493Q mutation, may have mitigated the effect of the N460K mutation.

[0304] Interestingly, BA.2.75 was more susceptible to Omi-32 (IGHV-3-33) than BA.2, with an 8-fold increase in neutralization titer, and the enhanced activity with Omi-32 is likely due to a stronger antibody-RBD interaction with the G446S mutation (Figure S19A, Table 22).

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

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

[0307] Escape from monoclonal antibodies by BA.2, BA.4, and BA.5 sublineages Reduced mAb activity was also observed in the emerging BA.2, BA.4 and BA.5 sublineages (including BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BQ.1, BQ.1.1, XBB, XBB.1 and XBB.1.5) (Figure 34), with XBB producing the most extreme escape. BA.2.75.2 reduced the activity of all nine IGHV3-53 / 66 mAbs by >100-fold, completely knocking out activity in 5 / 9. Only one mAb, Omi-42, was unaffected by all variants. Omi-42 is unusual in that it binds behind the left shoulder of the RBD, a region that remains untargeted by the emerging set of BA.2 variants (Nutalai et al., 2022), likely due to the relatively low number of antibodies that bind to this region.

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

[0309] Neutralization of BA.2 subtypes BA.2.11, BA.2.12, and BA.2.13 by vaccine sera The receptor binding capabilities of the BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 were also evaluated. A high-resolution crystal structure of the BA.2.12.1 RBD was generated, demonstrating differential sensitivity 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.

[0310] Considering the physicochemical properties of the side chain of residue 452, BA.2.13 is predicted to be a relatively modest change, going from L to M, which increases the size of the side chain but maintains hydrophobicity. BA.2.12.1 from L to Q introduces some polar character, while BA.2.11 is the most radical, from L to R introducing a large basic amino acid.

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

[0312] First, we observed neutralization profiles from sera collected 4 weeks after the third dose of the Oxford-AstraZeneca vaccine AZD1222 (n=41) or the Pfizer-BioNtech vaccine BNT162b2 (n=18). No significant loss of neutralization titers was observed compared to BA.2. In fact, BA.2.13 showed a significant increase (1.6-fold, p<0.0001) for AZD1222 vaccine recipients (Figure 27a, b). This is in contrast to a recent report (Cao, Y., et al., "BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection". Nature, 2022) in which sera collected from individuals who had received three vaccinations (4 weeks after the third vaccination of inactivated CoronaVac or after a ZF2001 booster vaccination after the second vaccination of CoronaVac) showed significantly reduced neutralizing titers for both BA.2.12.1 and BA.2.13 (BA.2.11 was not tested).

[0313] 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 We next examined the neutralization profiles of serum samples collected from vaccine recipients infected with BA.1. Samples (n=14) were collected 28 days or more after symptom onset (median 38 days). All convalescent individuals had received at least two vaccine doses, and three of them had received a third vaccine dose after Omicron infection. All three variants had significantly reduced neutralization titers compared to BA.2, with the greatest reduction in BA.2.11 (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) (Figure 27c). Taken together, these observations suggest that, compared to BA.2, its subvariants do not display stronger humoral immune escape in individuals vaccinated with three doses of AZD1222 or BNT162b2. However, in vaccinees with BA.1 breakthrough infection, broadly neutralizing antibody responses with high titers are elicited against all variants of concern, regardless of the vaccine administered, whereas BA.2 variants have a greater ability to evade humoral responses (Nutalai, R., et al., “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”. Cell, 2022. 185(12): p. 2116-2131 e18). This may suggest different selective pressures for BA.2 and its subvariants in the background of high breakthrough infection. As antibody titers naturally decay at longer time points, BA.1 breakthrough infected individuals are expected to be more susceptible to reinfection with BA.2 subvariants.

[0314] To further elucidate the differential responses between BA.2 and its subvariants, pseudovirus assays were performed with a panel of potent human monoclonal antibodies (mAbs) generated from a case of BA.1 breakthrough infection (Nutalai, et al., 2022). (Figure 29). Consistent with the structural observations and neutralization results, the greatest reduction in neutralization titers was seen with BA.2.11, followed by BA.2.12.1 and BA.2.13, with neutralization of BA.2.11 being completely knocked out by 5 / 27 mAbs (Omi-06, Omi-24, Omi-30, Omi-31, and Omi-34). Neutralizing activity against BA.2.12.1 was also reduced to various degrees for the same set of mAbs, while the profile against BA.2.13 remained largely unchanged. Of these, Omi-06 belongs to the IGVH4-4 family, while the other four mAbs belong to the IGVH1-69 family. Indeed, 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 effects were directly attributable to changes in RBD binding, we used surface plasmon resonance (SPR) 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 was reduced, with BA.2.12.1 RBD binding being 15-fold weaker than BA.2 for Omi-06, and remarkably, BA.2.12.1 RBD binding to Omi-31 being approximately 1300-fold weaker (Figure 30).

[0315] 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, compared to BA.2, healthy vaccine recipients who received three doses of the Oxford-AstraZeneca or Pfizer-BioNtech BNT162b2 vaccines do not appear to have increased humoral immune escape. This result differs from that of vaccine recipients who received three doses of the CoronaVac vaccine, where a significant decrease in neutralizing titers was observed (Cao, Y., et al., BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature, 2022). Nevertheless, vaccinees who experienced BA.1 breakthrough infections showed a significant reduction in neutralizing titers, regardless of which type of vaccine they received, which may be due in part to partial or complete knockout of neutralizing activity of antibodies belonging to the IGVH1-69 family, many of which are sensitive to mutations at leucine 452 in the spike RBD. This suggests that the continuously evolving omicron sublineage can evade the humoral immune response initiated by BA.1, and therefore suggests that the BA.1 spike or RBD may not be a significantly better immunogen than the ancestral Wuhan strain for the development of next-generation SARS-CoV-2 vaccines.

[0316] BA.2.12.1 Crystal structure The crystal structure of BA.2.12.1RBD was determined at 2.38 Å in a ternary complex with a neutralizing Fab and nanobody ( Figure 27 h–m ), showing that structural differences are essentially restricted to the side chain of residue 452.

[0317] Neutralization of BA.2.75.2 by mAbs generated after BA.1 infection Neutralization of BA.2.75.2 by a panel of mAbs generated after BA.1 infection (Nutalai et al., 2022) was investigated. A decrease in mAb activity against BA.2.75.2 was observed (Table 32a). BA.2.75.2 reduced the activity of all nine IGVH3-53 / 66 mAbs by more than 100-fold, completely knocking out activity in 4 / 9. Only one mAb, Omi-42, was unaffected by all variants and showed neutralization of the described BA.4+14 mutation with an IC50 of 11 ng / ml. Omi-42 is unusual in that it binds behind the left shoulder of the RBD, a region that remains untargeted by mutations (Nutalai et al., 2022), possibly due to the relative paucity of antibodies that bind to this region.

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

[0319] Serum neutralization titers of Pfizer BNT162b2 vaccine against BA.2.75.2 and BA.2.3.20. Neutralizing sera collected 28 days after the third dose of the Pfizer BNT162b2 vaccine ( Polack et al., 2020 ) and BA.4 / 5, BA.1, and BA.2-infected cases; characteristics of these subjects are described in Methods.

[0320] Using sera obtained 28 days after BNT162b, infectious titers against BA.2.75.2 were significantly reduced compared to BA.2 and BA.4, and were the lowest of all variants tested compared to the ancestral strain Victoria. The reduction in titers against BA.2.75.2 was in contrast to BA.2.75, which showed only a slight reduction compared to BA.2. Titers against BA.2.3.20 were also significantly reduced.

[0321] Using sera obtained after infection with BA.1, BA.2, and BA.4 / 5, neutralization titers of BA.2.75.2 and BA.2.3.20 were similarly significantly reduced compared to BA.4 / 5. Neutralization of the BA.4+14 RBD mutation described above was also reduced compared to BA.2 and BA.4 / 5, but not as significantly as BA.2.75.2, indicating a dominant effect of the BA.2.75.2 mutation.

[0322] Neutralization of BA4.6 by sera from vaccine breakthrough BA.1 or BA.2 infections Here, we study the neutralization profile of BA.4.6 using: Pfizer-BioNtech vaccine sera, BA.1, BA.2 and BA.4 / 5 vaccine breakthrough immune sera and a panel of monoclonal antibodies. Notably, we demonstrate further antibody evasion of BA.4.6, providing guidance for vaccine design and the use of therapeutic monoclonal antibodies.

[0323] To evaluate the antibody evasion ability of BA.4.6, 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. Figshare, 2020.), as well as Victoria, an early pandemic Wuhan-related strain used as a control. First, we examined the neutralization profile with sera collected 4 weeks after the third dose of the Pfizer-BioNtech vaccine BNT162b2 (n=22). Compared to BA.4 / 5, neutralization titers against BA.4.6 were reduced 2-fold in BNT162b2 sera (p<0.0001) (Figure 28a).

[0324] Neutralization profiles of serum samples collected from vaccine recipients infected with BA.1 were assayed. Samples (n=16) were collected ≥28 days after symptom onset. BA.2 samples (n=23) were collected ≥12 days after symptom onset or BA.4 / 5. Samples (n=11; all but one vaccinated) were collected >23 days after symptom onset (Figure 28b-d). Neutralization titers against BA.4.6 were significantly reduced compared to BA.4 / 5 for both breakthrough BA.1 (1.5-fold, P=0.0006) and BA.2 (1.2-fold, P=0.0384) serum samples. Notably, BA.4.6 was able to effectively escape neutralization by serum samples from BA.1 breakthrough infections, with significantly reduced titers compared to BA.1 (4.4-fold, p=0.0001), BA.2 (3-fold, p=0.0009) and BA.4 / 5 (1.5-fold, p=0.0006). 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.

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

[0326] Neutralization of BA.4.6 by mAbs in clinical use Finally, the neutralizing activity of some mAbs in clinical use was evaluated (Figure 28f). The potency of AZ1061 / silgavimab against BA.4 / 5 was completely knocked out against BA.4.6, resulting in a complete loss of activity of AZ7742 / Evasheld (the combination of AZ1061 / silgavimab and AZ8895 / tixagevimab, already inactive against BA.4 / 5). The activity of S309 / sotrviimab (no longer approved for COVID-19 treatment by the US Food and Drug Administration (FDA) since April 2022 due to its ineffectiveness against BA.2) was further reduced compared to BA.2 and BA.4 / 5. Thus, Ly-Cov1404 / bebuterovimab remains the only option for the treatment of BA.4.6.

[0327] In summary, BA.4.6 showed reduced neutralization by sera from 3-dose Pfizer vaccine recipients and BA.1 and BA.2 vaccine breakthrough recoveries compared to BA.4 / 5. Of note, BA.4.6 does not appear to be more resistant to neutralization by sera from BA.4 / 5 breakthrough infections compared to other variants. Taken together, these findings suggest that infection or breakthrough infection with BA.4.6 is likely to occur unless a person has recovered from BA.4 / 5 infection and has received 3 doses of the vaccine, which may provide some protection against BA.4.6.

[0328] As of September 2022, bivalent booster vaccinations combining ancestral and Omicron BA.1 are being rolled out in the UK and were recently approved by the FDA. It remains to be seen how effective these bivalent boosters are at preventing BA.4.6 infection. Finally, BA.4.6 further impaired the activity of Evershelled, which maintained activity against BA.4 / 5. As a result, currently only LY-CoV1404 bebterovimab retains efficacy against all circulating SARS-CoV-2 variants.

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

[0330] The effect on antibodies with broadly similar epitopes can be dramatically different, and this is equally true for antibodies with 452 or 486 in the center of their binding footprint. Thus, although Omi-31 (IGVH1-69) and Omi-32 (IGVH3-33) both bind in front of the right shoulder and their CDR-H3 is located near 452, the activity of Omi-31 is abolished by L452R (as detailed above) and Omi-32 is significantly enhanced (Figures 8A, 11A, B). Similarly, Omi-25 and Omi-42 both belong to the IGVH3-9 gene family and their footprints are in the 486 region (Figures 11C, D). Omi-25 contacts F486 and neutralization of BA.4 / 5 is suppressed. In contrast, Omi-42 does not contact either mutation site, and neutralization is fully retained for BA.4 / 5 (Fig. 10H,I, 11D).

[0331] Detailed mapping of RBD-antibody binding using competition assays. Using a matrix of pairwise BLI measurements, we mapped potent RBD-binding Omicron mAbs as well as several pre-pandemic mAbs with known binding locations.

[0332] This approach yielded consistent predictions. The mAbs segregate into a limited set of epitopes that appear to be a subset of those observed in early pandemic viruses and are quite distinct from the focus seen in beta. Essentially, the antibodies cluster into two regions, one containing VH3-53 and VH3-66 type antibodies located at the back of the neck / left shoulder, extending to the top of the left shoulder, and the other located at the front of the right shoulder of the neck, extending towards the known antibody binding site at S309. This region is dominated by VH1-69 family antibodies, with the exception of Omi-2, which is located within the other cluster. The mAb Omi-09, which shows reduced neutralization of beta and gamma, is located close to residue 484, mutated from Glu to Lys in beta / gamma and Ala in omicron. VH1-69 mAbs Omi-24, 30, 31, and 34, which show reduced neutralization of Delta, are located near residue 452, which is mutated from Leu to Arg in Delta.

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

[0334] Omi-3 belongs to the VH3-53 gene family and shows how this gene family can adapt to broadly neutralize all major SARS-CoV-2 variants (but does not bind to the SARS-CoV-1 RBD, like all potent omicron antibodies). The fundamental problem with these antibodies is that most VoCs have the mutation N501Y, which introduces a steric clash with the LC CDR1 (L1) that can abolish binding of the majority of VH3-53-containing antibodies. However, two mechanisms have been previously reported to move L1 to avoid this clash (Dejnirattisai et al., 2021b; Liu et al., 2021b). mAb-222, isolated from an individual infected with an early pandemic strain, has a proline inserted at residue 30 that can pack against Tyr-501 without a clash (Dejnirattisai et al., 2021b), which allows it to effectively neutralize alpha, beta and gamma variants. Beta27 uses an alternative mechanism, extending the HC CDR3 (H3) loop from the usual 9 to 11 residues and displacing L1, creating enough space to allow 501Y to be stabilized by backbone interactions that confer similar cross-reactivity ( Liu et al., 2021b ).

[0335] Omi-3 uses the same mechanism as beta-27 to accommodate the N501Y mutation, but Omi-3 H3 is again one more residue longer. Other VH3-53 omicron antibodies (Omi-18 and Omi-29) have a very similar H3 to beta-27 and likely use the same mechanism. This L1 configuration is also compatible with the Y505H mutation of omicron. However, neither 222 nor beta27 can effectively neutralize omicron, which may be due to certain features of the H3 loop that make close contact with the Q493R omicron mutation.

[0336] Omi-9 is one of three VH3-30 mAbs that bind across the left shoulder of the RBD. Omi-9 shows relatively weak neutralization of beta and gamma (Figure 2). Other antibodies with a high degree of sequence similarity bind similarly, with H3 contacting residue 484. Although the Omi-9 / BA.1 complex is at lower resolution (4.2 Å), it is clear that H3 contacts residue 484, explaining the sensitivity to E484K in beta and gamma, whereas E484A is tolerated in omicron.

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

[0338] In contrast, Omi-12 is adapted and able to potently neutralize omicrons and all VoC (Fig. 2A,B). VH1-58 antibodies bind to an epitope on the left shoulder and H3 contacts S477N, but mutations at this position in iota did not affect neutralization of VH1-58 mAbs using pseudovirus assays. Furthermore, mAb253 is still able to neutralize delta despite the T478K mutation. BA.2 of early pandemic mAb150 (VH3-53). Detectable residual activity was observed with BA.1, BA.1.1 and BA.2 (BA.3 was not tested). Two complex structures were obtained in different space groups, but these were very similar, providing three independent views of the complex. mAb150 binds in a pose similar to those previously observed for early pandemic viruses, but is translated and forms looser interactions, consistent with an almost complete loss of neutralizing activity. This demonstrates the dramatic impact of the adaptive mutations seen in Omi-3.

[0339] Interestingly, in BA.2, three serine residues mutated in the BA.1 RBD: 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 to Phe, meaning that it is likely a single point mutation from the early pandemic, whereas the S317L mutation in BA.1 requires two mutations. Thus, BA.2 may have a function in common with earlier versions of the Omicron lineage. Furthermore, the various views provided for this part of the structure show that it adopts constant, different conformations. This is likely due to different crystal contacts and reflects the flexibility of this loop region. This may have a biological function, as the Ser mutation requires a double codon change and may affect the presentation of the RBD. In the early pandemic viruses, VoC, Omicron BA.1 and BA.2, this loop can be observed from multiple views, confirming that flexibility is maintained across all variants.

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

[0341] Known antibodies C (AZD1061) and D (AZD8895): Known antibodies C (AZD1061) and D (AZD8895) bind to the back of the left shoulder and the front of the right shoulder, respectively, and both show reduced neutralization effects. Known antibody C (AZD1061) can still neutralize BA.2 and BA.3 (approximately 10-fold reduction), but neutralization of BA.1 is reduced by more than 100-fold compared to Victoria, and BA.1.1 is reduced by more than 1000-fold compared to Victoria. Known antibody C (AZD1061) is affected by contact with the G446S (absent in BA.2 and BA.3) and R346K (BA.1.1) mutations (L2 and H3 contact). Known antibody D (AZD8895) is a VH1-58 antibody that contacts residues 477 (H3) and 493 (H2) and is compromised by the S477N and Q493R mutations ubiquitously present in the Omicron lineage. Known antibody E (AZD7442) (a combination of C and D), as a sum of its components, maintains some neutralizing activity against Omicron strains.

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

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

[0344] Known Antibody K (S309): Known Antibody K (S309) retains appropriate activity across the Omicron lineage. S309 binds H3 on the right side, contacting the G339 and N343 glycans, the latter of which is in close proximity to the serine 371, 373, and 375 mutations. The S371F mutation in BA.2 (as opposed to S371L) may affect binding and result in slightly weaker activity against this virus.

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

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

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

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

[0349] Effects of mutations in BA.2.75 The set of mutations in BA.2.75 compared to BA.2 have opposing effects on neutralization. The reversion mutation R493Q makes it easier to neutralize the virus using vaccine sera (the vaccine contains Q493), whereas N460K significantly reduces neutralization titers when expressed alone compared to the combination of mutations seen in BA.2.75. N460K is a novel substitution that has not appeared in any previous variant of SARS-CoV-2. This mutation was introduced into the BA.2 backbone and its impact on neutralization by BNT162b2 sera was evaluated. Strikingly, BA.2+N460K titers were reduced 3.1-fold compared to BA.2, which was greater than the reduction seen with BA.2.75 and comparable to that seen with BA.4 / 5.

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

[0351] Although we were unable to express the BA.2+N460K RBD, previous studies using yeast display have shown that N460K can enhance ACE2 RBD binding, an effect similar to that seen with the N501Y mutation first described in alpha (Zahradnik et al., 2021). Thus, N460K can enhance antibody escape and increase receptor binding affinity.

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

[0353] These data suggest that a delicate balance may exist between antibody escape and ACE2 receptor affinity. Mutations in BA.2.75 result in reduced neutralizing titers of vaccine sera compared to BA.2. Although individual mutations in BA.2.75 may cause a large reduction in neutralizing titers compared to the full BA.2.75S sequence, these are balanced by the R393Q reversion mutation, which may have been selected to increase affinity for ACE2 and enhance transmissibility of BA.2.75.

[0354] Affinity of BA.2.11, BA.2.12, and BA.2.13 RBDs to ACE2 To assess possible changes in transmissibility of the BA.2 subvariants, SPR experiments were performed to analyze RBD binding to ACE2 (Figure 27d-g). The three RBD variants had an affinity of approximately 3 nM for ACE2, which was 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 increased affinity may result from a slightly improved complementarity between ACE2 and the RBD due to the mutation of leucine 452. Thus, these variants may have a slight advantage in transmissibility over BA.2.

[0355] Antigenic maps of BA.3 and BA.4 / 5 The neutralization data above have been used to place BA.3 and BA.4 / 5 on the antigenic map. The method used to analyze the delta and omicron variants was repeated (Liu et al., 2021, “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum”. Cell 184, 4220-4236 e4213), where each virus was modeled independently, allowing for serum-specific scaling of the response. The measured and modeled responses are shown in Figure 13A (1551 observations and 340 parameters, with a residual error of 23%). The results are best visualized in three dimensions (see the 2D projection in Figure 13B). This shows that, as expected, the omicron sublineages cluster together, but are well separated from the pandemic early viruses and early VoC. Within the omicron cluster, BA.4 / 5 are furthest from the pleomicron viruses.

[0356] Antigen map of BA.2.75 Neutralization of BA.2.75 was tested using sera from previously infected individuals over the course of the pandemic. These included sera obtained early in the pandemic (before the emergence of alpha) along with sera obtained after alpha, beta, gamma, delta, BA.1 and BA.2 infection (Figure 25). As expected, BA.2.75 neutralization titers were lower than for the homologous infecting strains (e.g., alpha sera against alphaviruses). Most striking, however, was the complete loss of BA.2.75 neutralization using delta sera (no samples achieved 50% neutralization at 1 / 20 dilution). However, cases vaccinated before or after delta infection had much higher titers against BA.2.75.

[0357] Using these data, BA.2.75 was placed on a 3D antigenic map (Figure 22A,B) using a method previously reported in Tuekprakhon et al., 2022. First all VoCs were included (Figure 22A). This showed that BA.2.75 was grouped with other Omicron viruses and separated into one hemisphere of the 3D plot. BA.2.75 appeared to be well separated from other Omicron sublineages, especially BA.4 / 5. It is also noteworthy that in the figure, BA.2.75 and Delta are located diametrically opposite each other, highlighting the antigenic distance between these two viruses. Due to the high dimensionality of the data, true distances may be distorted in a 3D projection and therefore were only calculated for Omicron and early pandemic viruses (but retaining the full serological information of each). The result is shown in Figure 22B, reproducing the main features of the full plot but allowing the Omicron sublineage to be more widely distributed in 3D space. Remarkably, during the clustered early pandemic and BA.2 / BA.3 pair fusion, the points were distributed as a bipyramid that maximized separation, consistent with antigenic escape being a key evolutionary driver.

[0358] Example 3. Examples of antibodies that can be generated by swapping light chains between antibodies derived from the same heavy chain V-gene As discussed in the detailed description above, antibodies derived from the same heavy chain V gene may have their light chains swapped to produce an antibody 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 characteristics when compared to the "parent" antibody.

[0359] Tables 4-12 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 are derived, along with their specificity for the RBD or NTD of the spike protein of SARS-CoV-2.

[0360] Example 4. Materials and Methods Virus strains 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 in Brazil, delta was a gift from Wendy Barclay and Thushan de Silva to the UK G2P Genotype to Phenotype Consortium, and omicron was cultured from a positive throat swab (IRAS Project ID: 269573, Ethics Ref: 19 / NW / 0730. Briefly, VeroE6 / TMPRSS2 cells (NIBSC) were cultured in 1% fetal bovine serum, 2 mM The cells were maintained at 37°C in the presence of 5% CO2 in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with Glutamax, 100IU / ml penicillin-streptomycin, 2.5ug / ml amphotericin B, and then inoculated with 200ul of swab fluid. 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. The virus titer was measured by focus-forming assay in Vero CCL-81 cells (ATCC).

[0361] Sequencing of Omicron isolates shows 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.

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

[0363] Virus-containing supernatants were harvested at 80% CPE and stored at -80°C after centrifugation at 3000 rpm at 4°C. Viral titers were measured by focus-forming assay in Vero cells. Victoria passage 5, alpha passage 2 and beta passage 4 strains, gamma passage 1, delta passage 3 and omicron passage 1 were sequenced to verify the absence of changes to the predicted spike protein sequence and furin cleavage site.

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

[0365] Serum from recipients of Pfizer vaccine Pfizer vaccine sera were obtained from volunteers who had received one or two doses of the BNT162b2 vaccine. Vaccine recipients were healthcare workers based at Oxford University Hospitals NHS Foundation Trust with no known previous SARS-CoV-2 infection and enrolled in the OPTIC study as part of the Oxford Translational Gastrointestinal Unit GI Biobank Study 16 / YH / 0247 [Yorkshire and Humber-Sheffield Research Ethics Committee (REC)], amended for this purpose on 8 June 2020. The study was carried out 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 for all participants enrolled in the study. Participants received two doses of 30 micrograms of Pfizer / BioNtech BNT162b2 mRNA vaccine administered intramuscularly after dilution (0.3 mL each) 17 to 28 days apart, with samples taken approximately 28 days (range 25 to 38), 180 days (range 178 to 221), and 270 days (range 243 to 273) after, and then approximately 28 days (range 25 to 56) after receiving a third "booster dose" of BNT162B2 vaccine. The average age of those vaccinated was 37 years (range 22 to 66), with 21 men and 35 women.

[0366] Plasma from early pandemic and alpha cases Participants from the first wave of SARS-CoV2 in the UK and with sequence confirmation of the B.1.1.7 lineage 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 Protocol for Severe Emerging Infectious Diseases [Oxford REC C, Ref: 13 / SC / 0149] and the Gastrointestinal Diseases in Oxford: COVID Substudy [Sheffield REC, Ref: 16 / YH / 0247]. Diagnosis was confirmed by report of symptoms consistent with COVID-19 and a positive test for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose / pharyngeal) swab tested in an accredited laboratory. Blood samples were collected after consent at least 14 days after symptom onset. Clinical information including disease severity (mild, severe or critical infection as per recommendations from the World Health Organization) and time between symptom onset and sample collection and age of participants were obtained for all individuals at the time of sample collection. Plasma / serum samples were heat inactivated and then aliquoted so that no more than three freeze-thaw cycles were performed for data generation.

[0367] Sera from beta, gamma, delta and BA.1 infected cases Beta and delta samples from UK infected cases were collected according to the Oxford Gastro-intestinal illness: COVID sub-study "Innate and adaptive immunity against SARS-CoV-2 in healthcare worker family and household members" protocol discussed above and approved by the University of Oxford Central University Research Ethics Committee. All individuals had sequence-confirmed beta / delta infection or PCR-confirmed symptomatic illness. These were present during isolation and 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 informed consent agreeing to the collection of their data 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 International Reference Laboratory for Coronaviruses (WHO) at FIOCRUZ as part of the national surveillance for coronaviruses and had approval from the FIOCRUZ Ethics Committee (CEP 4.128.241) to receive and analyze consecutive samples of suspected COVID-19 cases for virological surveillance. Clinical samples were shared with Oxford University, UK under MTA IOC FIOCRUZ 21-02.

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

[0369] Astrazeneca-Oxford vaccine trial procedures and sample processing Full details of the randomized controlled trials of ChAdOx1 nCoV-19 (AZD1222) were previously published (PMID:33220855 / PMID:32702298). These trials were registered with ISRCTN (15281137 and 89951424) and ClinicalTrials.gov (NCT04324606 and NCT04400838). A copy of the protocol was included in the previous publication (Folegatti et al., 2020, Lancet 396, 467-478.).

[0370] Data from vaccine volunteers who received two vaccinations are included in this example. Vaccine doses were 5×10 10 Patients received either 100 virions (standard dose; SD / SD cohort, n=21) or half a dose (low dose) as the first dose and the standard dose as the second dose (LD / SD cohort, n=4). The interval between the first and second doses ranged from 8 to 14 weeks. Blood samples were collected on the day of vaccination and at pre-specified days after vaccination, e.g., 14 and 28 days after booster immunization, and serum was isolated.

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

[0372] Plasmid construction and pseudotyped lentiviral particle generation Pseudotyped lentiviruses expressing SARS-CoV-2 S proteins from the ancestral strain (Victoria, S247R), BA.1, BA.1.1 and BA.2 were constructed as previously described (Nie, Jianhui, et al. “Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.” Emerging microbes & infections 9.1 (2020): 680-686., Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16 (2021): 4220-4236.), with some modifications. Briefly, synthetic codon-optimized SARS-CoV-2 BA.1 and BA.2 were custom synthesized by GeneArt (Thermo Fisher Scientific GENEART). The inserts and pcDNA3.1 vectors were cloned using Gibson assembly. The Victoria(S247R) construct was previously described in Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.” Cell 184.16(2021):4220-4236.To construct BA.1.1, the BA.1 construct was used as a template to PCR amplify the mutagenesis primers for R346K (R346K_F 5'-GTGTTCAATGCCACCAAATTCGCCAGCGTGTAC-3' and R346K_R 5'-GTACACGCTGGCGAATTTGGTGGCATTGAACAC-3') together with two primers for pcDNA3.1 vector (pcDNA3.1_BamHI_F 5'-GGATCCATGTTCCTGCTGACCACCAAGAG-3' and pcDNA3.1_Tag_S_EcoRI_R 5'-GAATTCTCACTTCTCGAACTGAGGGTGGC-3'), purified using QIAquick Gel Extraction Kit (QIAGEN), and subjected to Gibson assembly. All constructs were verified by Sanger sequencing after isolating the plasmids using QIAGEN Miniprep Kit (QIAGEN).

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

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

[0375] Pseudovirus neutralization test Details of the pseudovirus neutralization test have been 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. Briefly, the neutralization activity of potent monoclonal antibodies (mAbs) generated from donors recovered from Omicron and Beta infections and from donors infected early in the pandemic in the UK was performed against Victoria, Omicron-BA.1, BA.1.1, BA.2, BA.2.11, BA.2.12.1, BA.2.13, BA.3, BA.4.6, BA.4 / 5, BA.2.75 and BA.2+N460K. Four-fold serial dilutions of each mAb were incubated with pseudovirus particles for 1 h at 37 °C and 5% CO2. Stable HEK293T / 17 cells expressing human ACE2 were then added to the mixture at 1.5×104 cells / well. 48 hours after transduction, culture supernatants were removed and 50 μL of 1:2 Bright-Glo™ Luciferase Assay System (Promega, USA) in 1×PBS was added to each well. Reactions were incubated at room temperature for 5 min and firefly luciferase activity was measured using a CLARIOstar® (BMG Labtech, Ortenberg, Germany). mAb neutralization rates were calculated relative to controls. Probit analysis was used to estimate the value of the dilution that inhibited half the maximum of pseudotyped lentivirus infection (PVNT50).

[0376] To measure the neutralizing activity of convalescent plasma / serum samples or vaccine sera, three-fold serial dilutions of samples were incubated with pseudovirions for 1 h, applying the same strategy as for mAbs.

[0377] DNA manipulation Cloning was performed using a non-restrictive approach (Peleg and Unger, 2014). Mutagenic megaprimers were PCR amplified (KAPA HiFi HotStart ReadyMix, Roche, Switzerland, cat. no. KK3605), purified using NucleoSpin® gel and PCR Clean-up kits (Nacherey-Nagel, Germany, ref. no. 740609.50) and cloned into pJYDC1 (Adgene ID: 162458) (Zahradnik et al., 2021a). The parent pJYDC1 molecule was cleaved by DpnI treatment (1 h, NEB, USA, cat. R0176) and the reaction mixture was electroporated into E. coli Cloni® 10G cells (Lucigen, USA). The accuracy of the mutagenesis was verified by sequencing.

[0378] Cloning of spike and RBD Expression plasmids for wild-type and omicron BA.1 spike and RBD for BA.1 and BA.2 were constructed to encode the human codon-optimized sequences from BA.1 (EPI_ISL_6640917) and BA.2 (EPI_ISL_6795834.2). The constructs of wild-type and BA.1 spike and RBD plasmids were the same as those previously described (Dejnirattisai, Wanwisa, et al. "The antigenic anatomy of SARS-CoV-2 receptor binding domain." Cell 184.8 (2021): 2183-2200). Using a synthetic codon-optimized RBD fragment of BA.2 as a template, 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.

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

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

[0381] Production of RBD The RBD-encoding plasmid was transfected into Expi293F™ cells (ThermoFisher) by PEI and cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) for 4 days at 30° C. and 8% CO2. 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).

[0382] Production of BA.2.75 RBD Plasmids encoding the RBD were transfected into Expi293F™ cells (ThermoFisher) with PEI and cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 37°C for 1 day, followed by 30°C with 8% CO2 for 3 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). Conditioned media was diluted 1:2 in binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). The RBD was purified by His-tag binding on a 5 mL HisTrap Nickel column (GE Healthcare) followed by purification on a Superdex75 10 / 300GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.

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

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

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

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

[0387] To measure the binding kinetics between SARS-CoV-2 spike and ACE2, a CM5 sensor chip was used. The sensor chip was first activated by injecting an equal mixture of EDC and NHS (Cytiva) at 20uL / min for 300 seconds, followed by injection of 20ug / mL of spiked sample in 10mM sodium acetate pH 5.0 (Cytiva) at 10uL / min into the sample flow cell of the sensor chip, and finally by injection of 1.0M ethanolamine-HCl, pH 8.5 (Cytiva) at 20uL / min for 180 seconds. The reference flow cell was left blank. ACE2 was injected into two flow cells at a flow rate of 30μl / min in a range of five concentrations prepared by serial two-fold dilutions using a single cycle kinetics program. For background subtraction, running buffer was also injected using the same program.

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

[0389] A Protein A sensor chip (Cytiva) was used to measure the binding affinity of BA.4 / 5 RBD with mAb Omi-12. Ig Omi-12 was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over two flow cells at a flow rate of 30 μl / min in a range of seven concentrations prepared by serial two-fold dilutions. For background subtraction, running buffer was also injected using the same program. All data were fitted to a 1:1 binding model using Prism9 (GraphPad).

[0390] To compare the binding profiles of BA.2 and BA.4 / 5 RBDs of mAbs Omi-06 / Omi-25 / Omi-26, a Protein A sensor chip (Cytiva) was used. mAbs in IgG format were immobilized at similar levels (~350 RU) on the sample flow cells of the sensor chip. The reference flow cell was left blank. A single injection of RBD at 200 nM was performed on the two flow cells at a flow rate of 30 μl / min. For background subtraction, running buffer was also injected using the same program. Sensorgrams were plotted using Prism9 (GraphPad).

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

[0392] Protein A sensor chips (Cytiva) were used to measure the binding kinetics between BA.2.75 or BA.2+R493Q RBD and ACE2. ACE2-Fc was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected into two flow cells at a flow rate of 30 μl / min in a range of five concentrations prepared by serial two-fold dilutions using a single cycle kinetics program. For background subtraction, running buffer was also injected using the same program. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

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

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

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

[0396] To compare the binding profiles of BA.2 and BA.2.75 RBD of mAb Omi-36, SensorChip Protein A (Cytiva) was used. mAb Omi-36 in IgG format was immobilized on the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected once at 200 nM over the two flow cells at a flow rate of 30 μl / min. For background subtraction, running buffer was also injected using the same program. Sensorgrams were plotted using Prism9 (GraphPad).

[0397] IgG mAb and Fab production AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, Lilly, 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 as previously described (Nutalai et al., 2022), and antibodies were purified from the supernatant on protein A. Fabs were digested with papain from purified IgG using the Pierce Fab Preparation Kit (Thermo Fisher) according to the manufacturer's protocol.

[0398] Quantification and statistical analysis Statistical analysis is reported in Results and figure legends. Neutralization was measured by FRNT. Percentage of focus reduction was calculated and IC50 (FRNT50) was determined using the Probit program of the SPSS package. Wilcoxon matched-pairs signed rank test was used for analysis and two-sided P values ​​were calculated based on geometric mean values.

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

[0400] Crystals of the BA.1-RBD / Omi-12 / beta-54 complex were formed in Hampton Research PEGRx condition 1-46 containing 0.1 M sodium citrate tribasic dihydrate pH 5.0 and 18% (w / v) PEG 20000. The BA.1-RBD / Omi-12 / beta-54 complex was screened in 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 FabOmi-12 alone were formed in these conditions.

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

[0402] X-ray data collection, structure determination and refinement Diffraction data were collected at 100K on beamline I03 at the 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 mounted in loops and pre-frozen, and immersed for 1 s in cryoprotectant containing 25% glycerol and 75% mother liquor. Diffraction images of 0.1° rotations were recorded on an Eiger2 XE 16M detector (exposure time 0.018 s per image, beam size 80 × 20 μm, beam transmission 10%, wavelength 0.9762 Å). Data were indexed, integrated and scaled using the automated data processing program Xia2-dials (Winter, 2010, Journal of applied crystallography 43, 186-190; Winter et al., 2018, Acta Crystallogr D Struct Biol 74, 85-97). For each data set, 360° of data were collected from a single crystal.

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

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

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

[0406] Example 5 antibody structure The structure of the BA.1 RBD / Fab Omi-12 / Fab beta-54 ternary complex was determined at 5.5 Å resolution (Table 19, Figure 6A). Even though the BLI experiments did not reveal any significant competition for binding between the two Fabs, a slight clash was observed between them. The high-resolution structure of the uncomplexed Omi-12 fab (2.1 Å resolution, Table 19) was modeled into the complex electron density (Figure 6B, 6C). Superimposition of Fab253 onto Fab Omi-12 suggests that Q493R may clash with the H2 loop of Fab253, although in Omi-12 H2 adopts a slightly flattened conformation. This conformational change is due to antibody maturation via the somatic mutation V53P in the heavy chain variable region of Omi-12, which forms a stacking interaction with Y489 (Figure 6D).

[0407] Both Omi-12 and antibody 253 are derived from the germline heavy chain IGHV1-58. Interestingly, similar to antibody 253, the other antibodies derived from the germline heavy chain IGHV1-58 described herein, namely beta-47, beta-25, antibody 55, antibody 165 and antibody 318, also have a valine (V) at position 53 in the heavy chain variable region, i.e., 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 the IMGT numbering. Based on the data, modification of any of these antibodies by substitution of the valine at position 53 with a proline (i.e., V53P in absolute numbering and V58P in IMGT numbering) may result in an antibody that may be effective against Omicron.

[0408] Additionally, antibody AZD8895 (heavy chain variable region amino acid sequence provided in SEQ ID NO: 963, light chain variable region amino acid sequence provided in SEQ ID NO: 965) also derives from germline heavy chain IGHV1-58 (see, e.g., Nat Microbiol 6, 1233-1244 (2021)). AZD8895 has an isoleucine (I) at position 53 of the heavy chain variable region, which corresponds to position 58 according to IMGT numbering. Based on the data herein, modifying the heavy chain variable region AZD8895 (SEQ ID NO: 963) by replacing the isoleucine at position 53 with a proline (i.e., I53P) using absolute numbering, or with I58P using IMGT numbering, can result in an antibody that may be effective against omicron.

[0409] Thus, the data indicate that the VH1-58 antibody, when modified to include a proline at position 53 (corresponding to position 58 according to the IMGT numbering) of the heavy chain variable region, is particularly effective against Omicron.

[0410] ACE2 / BA.2.75 RBD structure To elucidate the molecular mechanism of high affinity, the structure of BA.2.75 RBD with ACE2 was determined by crystal structure analysis (following the method described in Example 4). As expected, the binding mode was essentially indistinguishable from that observed previously (Figure 20A), but outside the ACE2 footprint, there was a significant rearrangement, including rearrangement of the flexible RBD371-375 loop and alignment of part of the C-terminal 6xHis tag. Figure 20B shows a close-up view of the binding interface compared to the ACE2 / BA.2 RBD complex. In other complexes (with either R or Q in RBD493), K31 of ACE2 tends to be disordered, but in the BA.2.75 complex it is properly aligned, which may allow K31 to form a potential hydrogen bond with the glutamine side chain and increase the affinity of ACE2.

[0411] table

[0412] [Table 2]

[0413] [Table 3]

[0414] [Table 4]

[0415] [Table 5]

[0416] [Table 6]

[0417] [Table 7]

[0418]

Table 8

[0419]

Table 9

[0420]

Table 10

[0421]

Table 11

[0422]

Table 12

[0423]

Table 13

[0424]

Table 14

[0425]

Table 15

[0426]

Table 16

[0427]

Table 17

[0428]

Table 18

[0429]

Table 19

[0430]

Table 20

[0431]

Table 21

[0432]

Table 22

[0433]

Table 23

[0434]

Table 24

[0435]

Table 25

[0436]

Table 26

[0437]

Table 27

[0438]

Table 28

[0439]

Table 29

[0440]

Table 30

[0441]

Table 31

[0442]

Table 32

[0443]

Table 33

[0444]

Table 34

[0445]

Table 35

[0446]

Table 36

[0447]

Table 37

[0448]

Table 38

[0449]

Table 39

[0450] [Table 40]

[0451] [Table 41]

[0452] [Table 42]

[0453] [Table 43]

[0454] [Table 44]

[0455] [Table 45]

[0456] [Table 46]

[0457] Sequence Listing Amino acid sequences of the heavy and light chain variable regions of selected antibodies

[0458] [Table 47]

[0459] [Table 48]

[0460] [Table 49]

[0461] [Table 50]

[0462] [Table 51]

[0463] [Table 52]

[0464] [Table 53]

[0465] [Table 54]

[0466] [Table 55]

[0467] Nucleotide sequences of the heavy and light chain variable regions of selected antibodies

[0468] [Table 56]

[0469] [Table 57]

[0470] [Table 58]

[0471]

Table 59

[0472]

Table 60

[0473]

Table 61

[0474]

Table 62

[0475]

Table 63

[0476]

Table 64

[0477]

Table 65

[0478]

Table 66

[0479]

Table 67

[0480]

Table 68

[0481] [Table 69]

[0482] [Table 70]

[0483] [Table 71]

[0484] Amino acid sequence of CDR

[0485] [Table 72]

[0486] [Table 73]

[0487] [Table 74]

[0488] [Table 75]

[0489] [Table 76]

[0490] Amino acid sequences of the heavy and light chain variable regions of selected antibodies

[0491] [Table 77]

[0492] [Table 78]

[0493] [Table 79]

[0494] [Table 80]

[0495] [Table 81]

[0496] [Table 82]

[0497] [Table 83]

[0498] Nucleotide sequences of the heavy and light chain variable regions of selected antibodies

[0499] [Table 84]

[0500] [Table 85]

[0501] [Table 86]

[0502] [Table 87]

[0503] [Table 88]

[0504] [Table 89]

[0505] [Table 90]

[0506] [Table 91]

[0507] [Table 92]

[0508] [Table 93]

[0509] [Table 94]

[0510] [Table 95]

[0511] Amino acid sequence of CDR

[0512] [Table 96]

[0513] [Table 97]

[0514] [Table 98]

[0515] Amino acid sequences of the heavy and light chain variable regions of selected antibodies

[0516] [Table 99]

[0517] [Table 100]

[0518] [Table 101]

[0519] [Table 102]

[0520] [Table 103]

[0521] [Table 104]

[0522] Nucleotide sequences of the heavy and light chain variable regions of selected antibodies

[0523] [Table 105]

[0524] [Table 106]

[0525]

Table 107

[0526]

Table 108

[0527]

Table 109

[0528]

Table 110

[0529]

Table 111

[0530]

Table 112

[0531]

Table 113

[0532]

Table 114

[0533]

Table 115

[0534]

Table 116

[0535] Amino acid sequence of CDR

[0536] [Table 117]

[0537] [Table 118]

[0538] [Table 119]

[0539] [Table 120]

[0540] [Table 121]

[0541] SEQ ID NO:961 - Amino acid sequence encoded by IGHV1-58 germline V gene sequence MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAA

[0542] SEQ ID NO:962-AZD8895 (COV2-2196) heavy chain variable region nucleotide sequence Genbank:MT763531.1 [ka]

[0543] SEQ ID NO:963-AZD8895 (COV2-2196) Heavy chain variable region amino acid sequence: GenBank:QLI33947.1 [ka]

[0544] SEQ ID NO:964-AZD8895 (COV2-2196) Light chain variable region nucleotide sequence: GenBank:MT763532.1 [ka]

[0545] SEQ ID NO:965-AZD8895 (COV2-2196) Light chain variable domain amino acid sequence: GenBank:QLI33948.1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSRGWTFGQGTKVEIK

[0546] SEQ ID NO: 966 - Spike glycoprotein amino acid sequence of WIV04 isolate Genbank reference QHR63260.2 [ka]

[0547] SEQ ID NO:967 - Amino acid sequence encoded by germline IGLV kappa 3-20EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

Claims

1. An antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, (a) a heavy chain variable domain comprising the sequence of SEQ ID NO: 952; and (b) a light chain variable domain comprising the sequence of SEQ ID NO:

954. An antibody comprising:

2. The antibody of claim 1 , wherein the antibody comprises an Fc region.

3. The antibody of claim 1 , wherein the antibody comprises an IgG1 constant region.

4. The antibody of claim 2, wherein the Fc region comprises at least one modification that extends serum half-life.

5. The antibody of claim 4, wherein the Fc region comprises M252Y / S254T / T256E (YTE) mutations.

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

7. A polynucleotide encoding the light chain variable domain and the heavy chain variable domain of the antibody according to any one of claims 1 to 6.

8. A vector comprising one or more polynucleotides according to claim 7.

9. A host cell comprising one or more polynucleotides according to claim 7.

10. A host cell comprising one or more vectors of claim 8.

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

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

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

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

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

16. 10. The antibody of any one of claims 1 to 6 for use in a method for treating or preventing a disease or complication associated with SARS-CoV-2 infection or a disease or complication associated therewith, such as COVID-19.

17. 14. The pharmaceutical composition of claim 13 for use in a method for treating or preventing a disease or complication associated with SARS-CoV-2 infection or a disease or complication associated therewith, such as COVID-19.

18. The SARS-CoV-2 infection is caused by a SARS-CoV-2 strain of the alpha, beta, gamma, delta, or Omicron lineage, optionally wherein the strain of the Omicron lineage is Omicron BA.2.11, Omicron BA.2.12.1, Omicron BA.2.13, Omicron BA.2.3.20, Omicron BA.2.10.4, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.2.75, BA.2.75.2, Omicron BA.3, Omicron BA.4.6, Omicron BA.4 / 5, Omicron BJ.1, Omicron BS.1, Omicron BN.1, Omicron BF.7, Omicron BQ.1, Omicron BQ.

17. The antibody of claim 16, which is Omicron XBB.1.1, Omicron XBB and / or Omicron XBB.

1.

19. The SARS-CoV-2 infection is caused by a SARS-CoV-2 strain of the alpha, beta, gamma, delta, or Omicron lineage, optionally wherein the strain of the Omicron lineage is Omicron BA.2.11, Omicron BA.2.12.1, Omicron BA.2.13, Omicron BA.2.3.20, Omicron BA.2.10.4, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.2.75, BA.2.75.2, Omicron BA.3, Omicron BA.4.6, Omicron BA.4 / 5, Omicron BJ.1, Omicron BS.1, Omicron BN.1, Omicron BF.7, Omicron BQ.1, Omicron BQ.

18. The pharmaceutical composition of claim 17, wherein the compound is selected from the group consisting of omicron XBB.1.1, omicron XBB and / or omicron XBB.

1.

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

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