Patent Application
20240376178
The content of the electronically submitted sequence listing (Name: 2943_2240006_Seglisting_ST26; Size: 858,690 bytes; and Date of Creation: May 13, 2024) filed with the application is incorporated herein by reference in its entirety.
The invention relates to antibodies useful for the prevention, treatment and/or diagnosis of coronavirus infections, and diseases and/or complications associated with coronavirus infections, including COVID-19.
A severe viral acute respiratory syndrome named COVID-19 was first reported in Wuhan, China in December 2019. The virus rapidly disseminated globally leading to the pandemic with >200M confirmed infections and over 4.4M deaths in 12 months. The causative agent, SARS-CoV-2, is a beta coronavirus, related to SARS-CoV-1 and MERS coronaviruses, all of which cause severe respiratory syndromes.
Tremendous advances in our understanding of the disease and the virus have been made in the months since the identification of SARS-CoV-2 as the causative agent a of COVID-19. There are now a number of proven treatments including dexamethasone and Tocilizumab as well as monoclonal antibodies (mAbs), which have been shown to be effective when used in both prophylactic and therapeutic settings (Baum et al., 2020, Science 369, 1014-1018). Despite these advances, the pandemic is far from under control, leading to successive waves of infection.
Coronaviruses have four structural proteins: nucleocapsid, envelope, membrane and spike (S) proteins. The spike protein is the most prominent surface protein. It has an elongated trimeric structure and is responsible for engagement of target cells and triggering fusion of viral and host membranes. The spike protein from SARS-CoV-2 and SARS-CoV-1 both use angiotensin-converting enzyme 2 (ACE2) as a cell surface receptor. ACE2 is expressed in a number of tissues, including epithelial cells of the upper and lower respiratory tracts.
The S protein consists of two subunits, S1, which mediates receptor binding, and S2, responsible for viral and host cell membrane fusion. It is a dynamic structure capable of transitioning to a post-fusion state by cleavage between S1 and S2 following receptor binding or trypsin treatment. In some SARS-CoV-2 sequences a furin protease cleavage site is inserted between the S1 and S2 subunits, and a mutation of the cleavage site attenuates 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 receptor binding domain (RBD). While both regions are immunogenic, the RBD contains the interacting surface for ACE2 binding. Although usually packed down against the top of S2, RBDs can swing upwards to engage ACE2. Monoclonal antibodies (mAbs) recognize one or both of ‘up’ and ‘down’ conformations.
The S protein is relatively conserved between SARS-CoV-2 and SARS-CoV-1 (76%), but the RBD and NTD are less conserved (74% and 50% respectively) than the S2 domain (90%). Conservation with MERS-CoV and the seasonal human coronaviruses is much lower (19-21%). Overall, SARS-CoV-2 antibodies show limited cross-reactivity, even with SARS-CoV-1.
S is involved in viral attachment to target cells via the interaction of cell surface expressed ACE2 with the S receptor binding motif (otherwise known as the ACE-2 footprint), a 25 amino acid patch that lies at the tip of the receptor binding domain (RBD), in the S1 fragment of spike. Following attachment, cleavage of S releases S1, allowing a major conformational change in S2 exposing the hydrophobic fusion loop, to execute fusion of viral and host cell membranes, releasing the viral genome into the host cell cytoplasm to initiate viral replication. Analysis of large panels of mAbs generated from SARS-CoV-2 infected individuals reveals mAbs binding to multiple epitopes across S1 and S2. The majority of mAbs generated against the original strains of SARS-CoV-2, although able to bind S with high affinity, show little or no neutralizing activity. Genomic surveillance of SARS-CoV-2 has identified many thousands of mutations in structural and non-structural proteins. However, towards the end of 2020, viral variants were described that rapidly became the dominant strains locally and led to global spread and their designation of variants of concern (VoC).
Alpha (B.1.1.7) was first identified in the UK, with increased transmission. B.1.1.7 harbours 9 amino-acid changes in the spike, including N501Y in the ACE2 interacting surface. Beta (501Y.V2 also known as B.1.351) was first reported in South Africa. Gamma (P.1, 501Y.V2) was first reported in Brazil, which have 10 and 12 amino-acid changes in the spike protein, respectively. Delta was first reported from India and has now spread globally, causing outbreaks in a number of countries. Omicron BA.1 was first reported in late November 2021 in Southern Africa and spread around the world, becoming the dominant variant in many countries and almost completely displaced Delta. A succession of sub-lineages of Omicron have emerged, including BA.1.1, BA.2, BA.2.12.1, BA.2.75 and BA.4/5, which have outcompeted preceding strains to become regionally or globally dominant. Over 30 mutations are found in the Omicron S protein, including 15 substitutions in the RBD, leading to increased transmissibility (Suzuki et al., 2022 “Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.” Nature 603, 700-705) and widespread large reductions in neutralizing antibody titres (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).
Omicron BA.2 was reported at nearly the same time as BA. 1. The proportion of Omicron infections caused by BA.2 has been increasing in several countries and it became the dominant sub-lineage in Denmark and India.
BA.1.1, containing an additional R346K mutation in RBD, at one point accounted for about 40% of Omicron sequences globally, and about 35-60% in the UK and the USA (Iketani et al., 2022 “Antibody evasion properties of SARS-CoV-2 Omicron sublineages.” Nature 604, 553-556), but was soon outcompeted by BA.2. BA.2, which contains 8 unique substitutions in S, including 6 within the RBD, and lacks 13 mutations found in BA.1 (Nutalai et al., 2022), has become the dominant strain across the world as of August 2022. Recently, BA.2.12.1 has been identified in multiple countries, and caused a large regional outbreak in the North America (58% of the sequences as of May 25, 2022) (Del Rio and Malani, 2022, “COVID-19 in 2022—The Beginning of the End or the End of the Beginning?” JAMA 327, 2389-2390).
It is now becoming clear that BA.2 has a small transmission advantage against BA.1 although there is no evidence of increased disease severity. In early April 2022, two new Omicron lineages were reported from Gauteng in South Africa and designated BA.4 and BA.5. BA.4 and BA.5 (which have identical S sequences) became the dominant Omicron strains in Gauteng, fuelling a new wave of infection in South Africa.
Since June 2022, BA.4/5, which has both higher receptor binding affinity and a markedly enhanced escape from antibody responses (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) quickly spread from South Africa across the world and has now become the new globally dominant strain, with BA.5 in the ascendency in many regions. These variants (particularly BA.5) now account for the majority of sequenced cases in many countries.
In early May 2022, a new Omicron sub-lineage designated as BA.2.75 emerged in India. This strain has since spread to many countries including the UK, US, Australia, Germany and Canada. However, the true prevalence of BA.2.75 is difficult to determine as sequencing in many countries is patchy and has been greatly scaled back.
All of these variants contain multiple mutations in S and include changes in the RBD, NTD and in some cases the furin cleavage site between S1 and S2. The RBD mutations found in Alpha (N501Y), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y) and Delta (L452R, T478K) are located in or closely adjacent to the ACE2 interacting surface where they have the potential to modulate ACE2 interaction and disrupt the binding of neutralizing antibodies. Increased affinity of ACE2 interaction has been dominated for Alpha, Beta, Gamma and Delta (7, 19, 19, 2-fold, respectively) and may play a role in increasing viral transmissibility. Omicron contains an unprecedented number of mutations concentrated in the Spike (S) gene which carries 30 substitutions plus the deletion of 6 and insertion of 3 residues. Omicron BA.1 (RBD mutations of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H) contains unique mutations S371L, G446S and G496S and in some isolates R346K (BA.1.1), while BA.2 carries S371F, T376A, D405N and R408S. BA.3 contains no unique mutations relative to BA.1 and BA.2 and appears to be a fusion of the two, being BA.1-like at the N terminus and switching to become BA.2-like at the C-terminus from the mutation G496S.
BA.2.75 contains multiple mutational changes in the S protein compared to BA.2, including four substitutions in the NTD (W152R, F157L, I210V and G257S) and four in the RBD: D339H, G446S, N460K and R493Q.
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. These contain a single mutation of L452R, L452Q and L452M compared to the BA.2 Spike receptor-binding domain (RBD) respectively (
Considering the physico-chemical properties of the side chain of residue 452, BA.2.13 would be expected to be a relatively modest change; L to M will increase the size of the side chain but it remains hydrophobic. L to Q in BA.2.12.1 introduces some polar character, whilst BA.2.11 is the most radical with L to R introducing a large basic amino acid.
Further variants, BA.4 and BA.5, which have identical S sequences, appear to have evolved from BA.2. The sequences of BA.4 and BA.5 are highly related to the sequence of BA.2, but contain additional mutations. In particular, residues 69 and 70 of the NTD have been deleted (also found in Alpha, BA.1 and and BA.3) and they contain two additional substitutions in the RBD: L452R (also found in Delta) and F486V. Finally BA.4 and BA.5 lack the Q493R change seen in BA.1 and BA.2, reverting to Q493 as in the Victoria/Wuhan strain. When looking at the RBD, BA.4 and BA.5 have assembled mutations at all of the previously described positions in the VoC Alpha (N501Y), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y), Delta (L452, T478K), the only difference is E484A in BA.4 and BA.5 rather than E484K Beta and Gamma.
As of September 2022, a new variant related to BA.4/5, designated BA.4.6, has emerged and expanded in the United States where BA.5 dominates (87.5% prevalence as of 10th September 2022, tripling from less than 2% of sequences in early July 2022 to over 6% in mid-August 2022). Compared to BA.4/5, BA.4.6 contains two extra mutations in the Spike protein (S), R346T in the RBD and N658S in the C-terminal domain. The R346T mutation has raised concern for enhanced antibody evasion over BA.4/5, as the R346K mutation in BA.1.1 reduced serum neutralisation compared to BA.1 and impaired the activity of a number of monoclonal antibodies (mAbs) (Nutalai, et al., 2022). SARS-CoV-2 detection kits using monoclonal antibodies have also been developed. Examples include lateral flow tests by, e.g. Innova (SARS-CoV-2 Antigen Rapid Qualitative Test) and Quidel (Sofia 2 SARS Antigen FIA). However, these tests are reported to be highly inaccurate.
As of January 2023, further variants have emerged such as BQ.1 and XBB, which carry up to 8 additional RBD amino-acid substitutions compared to BA.2.
Structure function mapping of panels of monoclonal antibodies (mAbs) isolated from infected cases has led to considerable understanding of the antigenicity of S and mechanisms of neutralization. The majority of potent neutralizing antibodies bind at or in close proximity to the footprint of ACE2 and function by blocking ACE2 interaction, thereby preventing cellular attachment and infection. A second site of interaction of potent mAbs is in proximity to N-linked glycan at position N343, exemplified by S309, these antibodies do not block ACE2 interaction but may function to destabilize the S trimer. The third group of potent mAbs bind to the N-terminal domain in S1 and their mode of action is at present unclear. Another RBD epitope of potential interest lies outside of the ACE2 footprint and whilst mAbs binding here are not potent neutralizers they can nevertheless effectively protect in vivo (Huo et al., 2020; Sun et al., 2021; Yuan et al., 2020; Zhou et al., 2020).
Following BA.5 several new trends were observed in the evolution of Omicron: i) the emergence of ‘second generation’ BA.2 variants (including derivatives of BA.5)—variants with long phylogenetic branch lengths, multiple antigenic mutations and a lack of genetic intermediates, for example BA.2.75, BJ.1, BS.1, BA.2.10.4 and BA.2.3.20 (van der Straten et al. 2022. Immunity 55, 1725-1731) and ii) accelerated antigenic drift, seen both in BA.5 (Tuekprakhon et al., 2022) and within these second generation BA.2 lineages, notably 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) has produced XBB. Many of these variants show a large degree of convergent evolution in known antigenic RBD residues, and mutations lie in areas that may threaten the binding of neutralizing antibodies, leading to further escape from protection from infection afforded by vaccine or previous SARS-CoV-2 infection, including prior Omicron infection.
At present a number of lineages are growing rapidly from within both the BA.2 and BA.5 branches. Most striking 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 (which naturally contains 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); from the BA.2.75 branch (which naturally contains G446S, N460K and the reversion R493Q), BA.2.75.2 (R346T and F486V), BN.1 (R346T, K356T, F490S). There are also examples of several other second generation BA.2 variant lines such as BJ.1 (aka BA.2.10.1.1; R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (G446S, F486P, S494P and the R493Q reversion), BS.1 (BA.2.3.2.1; R346T, L452R, N460K, G476S), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and the Q493R reversion), and finally a BA.2.75×BJ.1 recombinant, XBB (which relative to BA.2 contains R346T, L368I, V445P, G446S, N460K, F486S, F490S).
These second-generation BA.2 variants have become dominant globally, with BQ.1 alone accounting for 50% of infections as of 27 Dec. 2022 (https://cov-spectrum.org/explore/World/AllSamples/Past6M) and XBB.1.5 (XBB.1+F486P) expanding rapidly in North America).
Outside the RBD the degree of convergent evolution is lesser but still present. Many of the second-generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to that seen in the VoCs, for example A˜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).
All currently approved SARS-CoV-2 vaccines are designed to induce antibody (and T-cell) responses to S and contain the S sequence found in the original Wuhan strain.
There is therefore particular concern as to whether the S mutations in the VoCs could cause immune escape, leading to vaccine failure or susceptibility to repeat infections in previously infected individuals.
The extensive mutational burden in Omicron S disrupts the activity of the majority of mAb binding to the three sites of binding of potent antibodies described above, the ACE-2 footprint, around the N343 glycan and the NTD. This leads to severe knock down or complete loss of the neutralizing capacity of serum from natural infection or vaccination, which has contributed to the increased transmissibility and explosive spread of Omicron.
It is an object of the invention to identify further and improved antibodies useful for preventing, treating and/or diagnosing coronavirus infections, and diseases and/or complications associated with coronavirus infections, including COVID-19, especially the Omicron variants of concern (VoCs) and as-yet-unidentified variants having further mutations in the ACE-2 footprint, RBD and/or NTD in the spike protein of SARS-CoV-2.
The inventors identified 28 human monoclonal antibodies (mAbs) recognizing the spike protein of SARS-CoV-2 (see Table 3). These antibodies showed potent neutralisation activity against SARS-CoV-2. Some of the Table 3 antibodies demonstrated potent neutralization effects that were broadly effective against the hCoV-19/Wuhan/WIV04/2019 strain, as well as SARS-CoV-2 strains from various lineages, such as Victoria (Wuhan+S247R), Alpha, Beta, Gamma, Delta, Omicron, including Omicron BA.2.11, Omicron BA.2.12.1, Omicron BA.2.13, Omicron Omicron BA.2.3.20, Omicron BA.2.10.4, Omicron BA.1, Omicron BA.1.1, Omicron BA.2, Omicron BA.2.75, BA.2.75.2, Omicron BA.3, Omicron BA.4.6, Omicron BA.4/5, Omicron BJ.1, Omicron BS.1, Omicron BN.1, Omicron XBB, and/or Omicron XBB.1 strains.
Many of the Table 3 mAbs used public V-genes (V-genes shared by the majority of the population). The inventors have previously shown that it is possible to generate further antibodies by swapping the light and heavy chains of the antibodies in Tables 1, 2 and 3 which are derived from the same public V-genes. Antibodies derived from the same public V-genes provided particularly useful mixed-chain antibodies.
In particular, the inventors found that antibodies Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42 were particularly effective at cross-neutralising SARS-CoV-2 strains Victoria, Alpha, Beta, Gamma, Delta and Omicron.
Accordingly, the invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the antibody comprises at least three CDRs of any one of the 28 antibodies in Table 3.
The invention provides an antibody capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the antibody comprises at least three CDRs of antibody Omi12, or of any one of the 27 antibodies in Table 3.
The invention also provides a combination of antibodies comprising two or more antibodies according to the invention.
The invention also provides a combination of antibodies 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 consist 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 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 1 or Table 2.
The 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.
The invention also provides a method for producing an antibody that is capable of binding to the spike protein of coronavirus SARS-CoV-2, comprising culturing the host cell of the invention and isolating the antibody from said culture.
The invention also provides a pharmaceutical composition comprising: (a) an antibody or a combination of antibodies of the invention, and (b) at least one pharmaceutically acceptable diluent or carrier.
The invention also provides an antibody, a combination of antibodies or a pharmaceutical composition of the invention, for use in a method for treatment of the human or animal body by therapy.
The invention also provides an antibody, a combination of antibodies or a pharmaceutical composition of the invention, for use in a method of treating or preventing coronavirus infection, or a disease or complication associated with coronavirus infection.
The invention also provides a method of treating or preventing coronavirus infection, or a disease or complication associated with coronavirus infection in a subject, comprising administering a therapeutically effective amount of an antibody, a combination of antibodies or the pharmaceutical composition of the invention, to said subject.
The invention also provides a method of identifying the presence of coronavirus, or a protein fragment thereof, in a sample, comprising (i) contacting the sample with an antibody or combination of antibodies of the invention, and (ii) detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates the presence of coronavirus, or a fragment thereof, in the sample.
The invention also provides a method of treating or preventing coronavirus infection, or a disease or complication associated therewith, in a subject, comprising identifying the presence of coronavirus according to the method of the invention, and treating the subject with the antibody or combination according to the invention, an anti-viral or an anti-inflammatory agent.
The invention also provides the use of an antibody, a combination of antibodies or a pharmaceutical composition of the invention, for preventing, treating and/or diagnosing coronavirus infection, or a disease or complication associated therewith.
The invention also provides the use of an antibody, a combination of antibodies or a pharmaceutical composition of the invention, for the manufacture of a medicament for treating or preventing coronavirus infection, or a disease or complication associated therewith.
An antibody of the invention specifically binds to the spike protein of SAR-CoV-2. In particular, it specifically binds to the S1 subunit of the spike protein, such as the receptor binding domain (RBD) or N-terminal domain (NTD).
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 the 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 the 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.
Table 3 lists 28 individual antibodies that were identified from recovered breakthrough Omicron SARS-CoV-2-infected patients, whom had already been received two doses of the Pfizer vaccine. Table 1 lists 42 individual antibodies that were 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 that were 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 host & microbe 30(1)(2021): 53-68]. The antibodies in Table 1 are also referred to herein with a pre-fix “COVOX”, e.g. COVOX-222. The antibodies in Table 2 are also referred to with a pre-fix “β”, e.g. “β50”. The antibodies in Table 3 are also referred to with a pre-fix “O”, e.g. “O02”. Tables 1 to 3 list the SEQ ID NOs for the heavy chain variable region and light chain variable region nucleotide and amino acid sequences, and the complementarity determining regions (CDRs) of the variable chains, of each of the antibodies.
The antibody in Table 3 may be selected from the group consisting of Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38. These antibodies were surprisingly found to retain strong neutralisation of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g. an IC50 of ≤0.1 μg/ml against all live strains tested).
The antibody in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28 and Omi 08. These antibodies were surprisingly found to retain strong neutralisation of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g. an IC50 of ≤0.05 μg/ml against all live strains tested).
The antibody in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39 and Omi42. These antibodies were surprisingly found to retain strong neutralisation of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g. an IC50 of ≤0.02 μg/ml against all live strainstested).
The antibody in Table 3 may be selected from the group consisting of: Omi03 and Omi12. These antibodies were surprisingly found to retain very strong neutralisation of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron (e.g. an IC50 of ≤0.01 μg/ml against all live strains tested).
The antibody 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 neutralisation of the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron.
Accordingly, in one embodiment, the antibody in Table 3 may be Omi03. Omi03 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi03 is derived from a IGHV3-53 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi03, and not the light chain of Omi03. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 695, 696 and 697, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi03 (i.e. SEQ ID NO: 692). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 692.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi03, and not the heavy chain of Omi03. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 698 and 700, respectively, and a CDRL2 having the amino acid sequence: GAS. 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.
In one embodiment, the antibody in Table 3 may be Omi12. Omi12 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi12 is derived from a IGHV1-58 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi12, and not the light chain of Omi12. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 735, 736 and 737, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi12 (i.e. SEQ ID NO: 732). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 732.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi12, and not the heavy chain of Omi12. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 738 and 740, respectively, and a CDRL2 having the amino acid sequence: GAS. 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.
In one embodiment, the antibody in Table 3 may be Omi02. Omi02 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi02 is derived from a IGHV1-69 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi02, and not the light chain of Omi02. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 685, 686 and 687, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi02 (i.e. SEQ ID NO: 682). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 682.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi02, and not the heavy chain of Omi02. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 688 and 690, respectively, and a CDRL2 having the amino acid sequence: GAS. 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.
In one embodiment, the antibody in Table 3 may be Omi08. Omi08 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1 Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
In one embodiment, the antibody in Table 3 may be Omi42. Omi42 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi42 is derived from a IGHV3-9 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi42, and not the light chain of Omi42. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 955, 956 and 957, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi42 (i.e. SEQ ID NO: 952). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 952.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi42, and not the heavy chain of Omi42. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 958 and 960, respectively, and a CDRL2 having the amino acid sequence: EVS. 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.
In one embodiment, the antibody in Table 3 may be Omi16. Omi16 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi16 is derived from a IGHV3-66 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi16, and not the light chain of Omi16. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 745, 746 and 747, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi16 (i.e. SEQ ID NO: 742). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 742.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi16, and not the heavy chain of Omi16. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 748 and 750, respectively, and a CDRL2 having the amino acid sequence: GAS. 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.
In one embodiment, the antibody in Table 3 may be Omi18. Omi18 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi18 is derived from a IGHV3-53 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi18, and not the light chain of Omi18. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 765, 766 and 767, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi18 (i.e. SEQ ID NO: 762).
The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 762.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi18, and not the heavy chain of Omi18. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 768 and 770, respectively, and a CDRL2 having the amino acid sequence: DDS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 764.
In one embodiment, the antibody in Table 3 may be Omi20. Omi20 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi20 is derived from a IGHV3-66 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi20, and not the light chain of Omi20. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 775, 776 and 777, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi20 (i.e. SEQ ID NO: 772). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 772.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi20, and not the heavy chain of Omi20. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 778 and 780, respectively, and a CDRL2 having the amino acid sequence: AAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 774.
In one embodiment, the antibody in Table 3 may be Omi23. Omi23 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi23 is derived from a IGHV4-31 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi23, and not the light chain of Omi23. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 785, 786 and 787, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi23 (i.e. SEQ ID NO: 782). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 782.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi23, and not the heavy chain of Omi23. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 788 and 790, respectively, and a CDRL2 having the amino acid sequence: AAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 784.
In one embodiment, the antibody in Table 3 may be Omi28. Omi28 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi28, and not the heavy chain of Omi28. For example, the antibody may
In one embodiment, the antibody in Table 3 may be Omi39. Omi39 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
In one embodiment, the antibody in Table 3 may be Omi17. Omi17 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi17 is derived from a IGHV3-66 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi17, and not the light chain of Omi17. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 755, 756 and 757, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi17 (i.e. SEQ ID NO: 752). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 752.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi17, and not the heavy chain of Omi17. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 758 and 760, respectively, and a CDRL2 having the amino acid sequence: GAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 754.
In one embodiment, the antibody in Table 3 may be Omi29. Omi29 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi29 is derived from a IGHV3-53 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi29, and not the light chain of Omi29. For example, the antibody may comprise a CDRH1,CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 845, 846 and 847, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi29 (i.e. SEQ ID NO: 842). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 842.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi29, and not the heavy chain of Omi29. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 848 and 850, respectively, and a CDRL2 having the amino acid sequence: GAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 844.
In one embodiment, the antibody in Table 3 may be Omi36. Omi36 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
In one embodiment, an antibody of the invention may comprise a heavy chain variable domain and a light chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity to the heavy chain variable domain and light chain variable domain, respectively, of antibody Omi36 (i.e. SEQ ID NOs: 912 and 914, respectively). The heavy chain domain of Omi36 is derived from a IGHV3-66 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi36, and not the light chain of Omi36. For example, the antibody may comprise a 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.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi36, and not the heavy chain of Omi36. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 918 and 920, respectively, and a CDRL2 having the amino acid sequence: GAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 914.
In one embodiment, the antibody in Table 3 may be Omi38. Omi38 was found to neutralise the live SARS-CoV-2 variant strains Victoria, Alpha, Beta, Gamma, Delta and Omicron, and the psuedoviral constructs of Victoria, Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and Omicron BA.3 (see Tables 13 and 14,
The heavy chain domain of Omi38 is derived from a IGHV1-69 v-region, and the inventors have previously demonstrated that switching of the heavy chains and light chains between antibodies derived from the same v-region results in an antibody that is particularly useful with the invention (explained further below). Hence, an antibody of the invention may comprise the heavy chain of Omi38, and not the light chain of Omi38. For example, the antibody may comprise a CDRH1, CDRH2 and CDRH3 having the amino acid sequences specified in SEQ ID NOs: 925, 926 and 927, respectively. The antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of antibody Omi38 (i.e. SEQ ID NO: 922). The antibody may comprise a heavy chain variable domain comprising or consisting of SEQ ID NO: 922.
Alternatively, in an embodiment of the invention, the antibody may comprise the light chain of Omi38, and not the heavy chain of Omi38. For example, the antibody may comprise a CDRL1 and CDRL3 having the amino acid sequences specified in SEQ ID NOs: 928 and 930, respectively, and a CDRL2 having the amino acid sequence: DAS. 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 alight chain variable domain comprising or consisting of SEQ ID NO: 924.
An antibody of the invention may comprise a light chain variable domain comprising CDRL1, CDRL2 and CDRL3 from a first antibody in Table 1, 2 or 3 and a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 from a second antibody in Table 1, 2 or 3, with the proviso that the first and second antibodies are different. Such antibodies are referred to as mixed chain antibodies herein.
Examples of the mixed chain antibodies useful with the invention are provided in Tables 4 to 12. Table 4 shows examples of mixed chain antibodies generated from antibodies in Tables 1 to 3 that are derived from the same germline heavy chain IGHV 3-53. Table 5 shows examples of mixed chain antibodies generated from antibodies in Tables 1 to 3 that are derived from the same germline heavy chain IGHV 3-53 and IGHV3-66. Table 6 shows examples of mixed chain antibodies generated from antibodies in Tables 1 to 3 that are 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 that are derived from the same germline heavy chain IGHV1-69. Table 8 shows examples of mixed chain antibodies generated from antibodies in Tables 1 to 3 that are 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 that are derived from the same germline heavy chain IGHV3-33. Table 10 shows examples of mixed chain antibodies generated from antibodies in Tables 1 to 3 that are 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 that are 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 that are derived from the same germline heavy chain IGHV4-31. Examples of mixed chain antibodies that are derived from the same germline heavy chain IGHV1-69 are Omi02H/Beta-49L and Omi38H/Omi24L.
Hence, 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, with the proviso 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 the 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 the light chain variable domain from a second antibody in Table 1, 2 or 3, with the proviso 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 the 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 the light chain variable domain of an antibody in Table 1, 2 or 3, with the proviso that the first and second antibodies are different.
The first antibody may be in Table 3 and the second antibody may be in Table 3.
The first antibody may be in Table 3 and the second antibody may be in Table 1. The first antibody may be in Table 3 and the second antibody may be in Table 2. The first antibody may be in Table 1 and the second antibody may be in Table 3. The first antibody may be in Table 2 and the second antibody may be in Table 3. The first antibody may be in Table 1 and the second antibody may be in Table 2. The first antibody may be in Table 2 and the second antibody may be in Table 1. The first antibody may be in Table 2 and the second antibody may be in Table 2. The first antibody may be in Table 1 and the second antibody may be in Table 1.
In one embodiment, at least one of the first and second antibodies is an antibody from Table 3.
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 an antibody in Table 1 or 2.
In one embodiment, at least one of the heavy chain variable domain and the light chain variable domain are from Table 3.
The antibody in Table 3 may be selected from the group consisting of: Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42. The antibody in Table 3 may be selected from the group consisting of: Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 and Omi38. For example, the antibody in Table 3 may be selected from the group consisting of Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28 and Omi08. The antibody 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.
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 domain of these antibodies are derived from IGHV3-53. The resulting mixed chain antibodies are set out in Table 4. Hence, the 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% sequence identity to the corresponding variable domain of any one of the mixed chain antibodies as set out in Table 4.
Antibodies derived from IGHV3-53 may be used to produce mixed-chain antibodies with antibodies from IGHV3-66 (e.g. antibodies 40 and 398 in Table 1) (see, e.g. Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184(8) (2021): 2183-2200; Supasa, Piyada, et al. “Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.” Cell 184(8) (2021): 2201-2211; Zhou, Daming, et al. “Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.” 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)). Accordingly, 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 domain of these antibodies are derived from IGHV3-53 and IGVH3-66. The resulting mixed chain antibodies are set out in Table 5. Hence, the 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 as set out in Table 5.
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 domain of these antibodies are derived from IGHV 1-58. The resulting mixed chain antibodies are set out in Table 6. Hence, the 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 as set out in Table 6.
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 domain of these antibodies are derived from IGHV 1-69. The resulting mixed chain antibodies are set out in Table 7. Hence, the 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 as set out in Table 7.
In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: Beta-22, Beta-29, antibody 159, and Omi09. The heavy chain variable domain of these antibodies are derived from IGHV 3-30. The resulting mixed chain antibodies are set out in Table 8. Hence, the 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 as set out in Table 8.
In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: Beta-20, Beta-43, Omi32 and Omi33. The heavy chain variable domain of these antibodies are derived from IGHV 3-33. The resulting mixed chain antibodies are set out in Table 9. Hence, the antibody of the invention may comprise all six CDRs (CDRH1-3 and CDRL1-3), and/or a heavy chain variable domain and alight 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 as set out in Table 9. The CDRL1-3 of Omi32 and Omi33 are identical, meaning that they are, effectively, already exemplary mixed-chain antibodies of the invention.
In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: antibody 278, Beta-44, Omi26 and Omi41. The heavy chain variable domain of these antibodies are derived from IGHV 1-18. The resulting mixed chain antibodies are set out in Table 10. Hence, the 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 as set out in Table 10.
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 domain of these antibodies are derived from IGHV 3-9. The resulting mixed chain antibodies are set out in Table 11. Hence, the 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 as set out in Table 11.
In one embodiment, the first antibody and the second antibody are both selected from the group consisting of: Beta-56 and Omi23. The heavy chain variable domain of these antibodies are derived from IGHV 4-31. The resulting mixed chain antibodies are set out in Table 12. Hence, the 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 as set out in Table 12.
The constant region domains of an antibody molecule of the invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant regions are of human origin. In particular, human IgG (i.e. IgGI, IgG2, IgG3 or IgG4) constant region domains may be used. Typically, the constant region is a human IgGI constant region.
The invention also provides an antibody which is a full length antibody of any one of the antibodies in Tables 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In other words, an antibody of the invention comprises a heavy chain variable domain and a light chain variable domain consisting of the heavy chain variable domain and 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 a IgG (e.g. IgG1) constant region.
For example, the antibody of the invention may be a full length Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 or Omi42 antibody. The antibody of the invention may be a full length Omi03, Omi12, Omi02, Omi39, Omi42, Omi16, Omi18, Omi20, Omi 23, Omi28, Omi08, Omi17, Omi29, Omi36 or Omi38 antibody. These antibodies are all highly potent neutralising mAbs that have been shown to neutralise the Omicron variant of SARS-CoV-2 with an IC50 of ≤0.1 μg/ml. The antibodies also retain neutralisation of inter alia at least the Victoria, Alpha, Beta, Gamma and Delta strains of SARS-CoV-2 with an IC50 of ≤0.1 μg/ml.
The antibody may be derived from germline heavy chain IGHV1-58 and comprises proline at position 53 in the heavy chain variable region (according to absolute numbering). For example, the antibody may comprise a heavy chain variable domain comprising or consisting of an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of Omi-12 (SEQ ID NO: 731), Beta-47 (SEQ ID NO: 591), Beta-25 (SEQ ID NO: 461), antibody 55 (SEQ ID NO: 62), antibody 165 (SEQ ID NO: 182), antibody 253 (SEQ ID NO: 262), or antibody 318 (SEQ ID NO: 332), with the proviso that the amino acid at position 53 in the heavy chain variable region is proline (according to absolute numbering). For example, the antibody may comprise the heavy chain variable region and the light chain variable region 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 NO: 182 and 181, respectively), antibody 253 (SEQ ID NOs: 262 and 261, respectively), or antibody 318 (SEQ ID NOs: 332 and 331, respectively), except with a V53P mutation in the heavy chain variable region. The inventors found that such antibodies are particularly effective against Omicron strains (e.g. see Example 5).
The position 53 in the heavy chain variable region of the IGHV1-58-derived antibodies Omi-12, Beta-47, Beta-25, antibody 55, antibody 165, antibody 253, and antibody 318 corresponds to position 58 according to IMGT numbering.
Accordingly, the invention also provides an antibody derived from germline heavy chain IGHV1-58, capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein the amino acid at position 58 in the heavy chain variable region according to IMGT numbering is proline or is substituted with proline.
The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the heavy chain variable domain of an antibody derived from germline heavy chain IGHV1-58, with the proviso that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline.
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 Omi-12, Beta-47, Beta-25, antibody 55, antibody 165, antibody 253, or antibody 318 is described herein (e.g. see Tables 1 to 3). The amino acid sequence of the heavy chain variable domain of antibody AZD8895 is provided in SEQ ID NO: 963.
The IGHV1-58 germline V-gene sequence encodes the amino acid sequence: MQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGS GNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAA (SEQ ID NO: 961). Hence, the 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 IMGT numbering is proline or is substituted with proline.
The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963, with the proviso that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline. The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 591 461, 62, 182, 262, or 332, wherein the valine at position 58 according to IMGT numbering is substituted with proline.
The antibody may comprise a heavy chain variable domain comprising an amino acid sequence having SEQ ID NO: 963, wherein the isoleucine at position 58 according to IMGT numbering is substituted with proline.
In some embodiments, the antibody derived from germline heavy chain IGHV1-58, comprises a light chain variable domain derived from IGLV Kappa 3-20. The antibody may comprise a light chain variable domain comprising an amino acid sequence having ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the light chain variable domain of an antibody derived from germline IGLV Kappa 3-20. The germline IGLV Kappa 3-20V sequence may encode the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO: 967). Hence, the antibody derived from germline heavy chain IGHV1-58 may comprise a light chain variable domain comprising an amino acid sequence having ≥60%, ≥70%, ≥80%,_≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to SEQ ID NO: 967.
The 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 IMGT numbering is proline or is substituted with proline. The modified version of SEQ ID NO: 961 may comprise a modification as described herein, e.g. a substitution, deletion and/or addition. For example, the modification may comprise ≤50, ≤45, ≤40, ≤35, ≤30, ≤25, ≤20, ≤15, ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or 1 amino acid substitutions and/or deletions from SEQ ID NO: 961. The modification may comprise ≤4, ≤3, ≤2, or 1 amino acid substitutions and/or deletions from SEQ ID NO: 961.
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 which comprises ≤10, ≤9, ≤8, ≤7, ≤6, ≤5, ≤4, ≤3, ≤2, or 1 modifications, with the proviso that the amino acid at position 58 according to IMGT numbering is proline or is substituted with proline. The modified version of SEQ ID NO: 731, 591, 461, 62, 182, 262, 332 or 963 may comprise a modification as described herein, e.g. a substitution, deletion and/or addition.
The antibody may comprise a IgG (e.g. IgG1) constant region.
The invention also provides a method of preparing such antibodies. For example, the method may comprise modifying an antibody derived from the 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 in the heavy chain variable region (according to IMGT numbering) with proline. The antibody derived from the 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 these antibodies is described herein (e.g. see Tables 1 to 3 and SEQ ID NO: 963). The invention also provides an antibody obtainable or obtained by the method.
An antibody of the invention may be or may comprise a modification from the amino acid sequence of an antibody in Tables 1 to 12, whilst maintaining the activity and/or function of the antibody. The modification may a substitution, deletion and/or addition. For example, the modification may comprise 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 to 12. For example, the modification may comprise an amino acid substituted with an alternative amino acid having similar properties. Some properties of the 20 main amino acids, which can be used to select suitable substituents, are as follows:
The modification may comprise a derivatised amino acid, e.g. a labelled or non-natural amino acid, providing the function of the antibody is not significantly adversely affected.
Modification of antibodies of the invention as described above may be prepared during synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
Antibodies of the invention may be modified (e.g. as described above) to improve the potency of said antibodies or to adapt said antibodies to new SARS-CoV-2 variants. The modifications may be amino acid substitutions to adapt the antibody to substitutions in a virus variant. For example, the known mode of binding of an antibody to the spike protein (e.g. by crystal structure determination, or modelling) may be used to identify the amino acids of the antibody that interact with the substitution in the virus variant. This information can then be used to identify possible substitutions of the antibody that will compensate for the change in the epitope characteristics. For example, a substitution ofa hydrophobic amino acid in the spike protein to a negatively changes amino acid may be compensated by substituting the amino acid from the antibody that interacts with said amino acid in the spike protein to a positively charged amino acid. Methods for identifying residues of an antibody that may be substituted are encompassed by the present disclosure, for example, by determining the structure of antibody-antigen complexes as described herein.
The antibodies of the invention may contain one or more modifications to increase their cross-lineage neutralisation property. For example, E484 of the spike protein, which is a key residue that mediates the interaction with ACE2, is mutated in some SARS-CoV-2 strains (e.g. Victoria strain which contains E484, but P.1 and B.1.351 strains contain E484K) resulting in differing neutralisation effects of the antibodies. Thus, antibodies that bind to E484 can be modified to compensate for the changes in E484 of the spike protein. For example, E484 is mutated from a positively charge to negatively charged amino acid in SAR-CoV-2 strains of B.1.351 or P.1 lineage, when compared to the original strain. The amino acid residues of antibodies that bind to or near E484 may be mutated to compensate for the change in charge. Examples of such amino acid residues may be G104 and/or K108 in SEQ ID NO: 102 of antibody 88, or R52 in SEQ ID NO: 372 of antibody 384.
Antibodies of the invention may be isolated antibodies. An isolated antibody is an antibody which is substantially free of other antibodies having different antigenic specificities.
The term ‘antibody’ as used herein may relate to whole antibodies (i.e. comprising the elements of two heavy chains and two light chains inter-connected by disulphide bonds) as well as 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 (immunoreacts with) an antigen. By “specifically binds” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and at least one heavy chain constant region. Each light chain is comprised 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 a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
Antibodies May Include, but are not Limited to, Polyclonal, Monoclonal, Chimeric, dAb (Domain Antibody), Single Chain, Fab, Fab′ and F(Ab′)2 Fragments, scFvs, and Fab Expression Libraries
An antibody of the invention may be a monoclonal antibody. Monoclonal antibodies (mAbs) of the invention may be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example those disclosed in “Monoclonal Antibodies: a manual of techniques” (Zola H, 1987, CRC Press) and in “Monoclonal Hybridoma Antibodies: techniques and applications” (Hurrell J G R, 1982 CRC Press). An antibody of the invention may be multispecific, such as bispecific. A bispecific antibody of the invention binds two different epitopes. The epitopes may be in the same protein (e.g. two epitopes in spike protein of SARS-CoV-2) or different proteins (e.g. one epitope in spike protein and one epitope in another protein (such as coat protein) of SARS-CoV-2).
In one embodiment, a 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 to the RBD of the spike protein. The bispecific antibody may bind to two different epitopes in the RBD of the spike protein.
One or more (e.g. two) antibodies of the invention can be coupled to form a multispecific (e.g. bispecific) antibody. Methods to prepare multispecific, e.g. bispecific, antibodies are well known in the art.
An antibody may be selected from the group consisting of single chain antibodies, single chain variable fragments (scFvs), variable fragments (Fvs), fragment antigen-binding regions (Fabs), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigen-binding domain of a native antibody or an aptamer, single-domain antibodies (sdAbs), also known as VHH antibodies, nanobodies (Camelid-derived single-domain antibodies), shark IgNAR-derived single-domain antibody fragments called VNAR, diabodies, triabodies, Anticalins, aptamers (DNA or RNA) and active components or fragments thereof.
The constant region domains of an antibody molecule of the invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant regions are of human origin. In particular, human IgG (i.e. IgG1, IgG2, IgG3 or IgG4) constant region domains may be used. Typically, the constant region is a human IgGI constant region.
The light chain constant region may be either lambda or kappa.
Antibodies of the invention may be mono-specific or multi-specific (e.g. bi-specific). A multi-specific antibody comprises at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or to a different epitope on the same antigen.
An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody. Typically, the antibody is a human antibody. Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.
The antibody of the invention may be a full-length antibody.
The antibody of the invention may be an antigen-binding fragment. An antigen-binding fragment of the invention binds to the same epitope of the parent antibody, i.e. the antibody from which the antigen-binding fragment is derived. An antigen-binding fragment of the invention typically retains the parts of the parent antibody that interact with the epitope. The antigen-binding fragment typically comprise the complementarity-determining regions (CDRs) that interact with the antigen, such as one, two, three, four, five or six CDRs. In some embodiments, the antigen-binding fragment further comprises the structural scaffold surrounding the CDRs of the parent antibody, such as 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.
An antigen-binding fragment does not necessarily have an identical sequence to the parent antibody. In one embodiment, the antigen-binding fragment may have ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity with the respective CDRs of the parent antibody. In one embodiment, the antigen-binding fragment may have ≥70%, ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, 100% sequence identity with the respective variable region domains of the parent antibody. Typically, the non-identical amino acids of a variable region are not in the CDRs.
The antigen-binding fragments of antibodies of the invention retain the ability to selectively bind to an antigen. Antigen-binding fragments of antibodies include single chain antibodies (i.e. a full-length heavy chain and light chain); Fab, modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv.
An antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma R et al., 1998, J. Immunol. Methods, 216, 165-181).
Methods for screening antibodies of the invention that do not share 100% amino acid sequence identity with one of the antibodies disclosed herein, that possess the desired specificity, affinity and functional activity include the methods described herein, e.g. enzyme linked immunosorbent assays, biacore, focus reduction neutralisation assay (FRNT), and other techniques known within the art.
With regards to function, an antibody of the invention may be able to neutralise at least one biological activity of SAR-CoV-2 (a neutralising antibody), particularly to neutralise virus infectivity.
Neutralisation may also be determined using IC50 or IC90 values. For example, the antibody may have an IC50 value of ≤0.1 μg/ml, ≤0.05 μg/ml, ≤0.01 μg/ml≤0.005 μg/ml or ≤0.002 μg/ml. In some instances an antibody of the invention may have an IC50 value of between 0.0001 μg/ml and 0.1 μg/ml, sometimes between 0.0001 μg/ml and 0.05 μg/ml or even between 0.0001 μg/ml and 0.001 μg/ml.
For example, the IC50 values of some of the antibodies of Tables 1 to 12 are provided in Tables 13 to 16.
The ability of an antibody to neutralise virus infectivity may be measured using an appropriate assay, particularly using a cell-based neutralisation assay, as shown in the Examples. For example, the neutralisation ability may be measured in a focus reduction neutralisation assay (FRNT) where the reduction in the number of cells (e.g. human cells) infected with the virus (e.g. for 2 hours at 37° C.) in the presence of the antibody is compared to a negative control in which no antibodies were added.
An antibody of the invention may block the interaction between the spike protein of SAR-CoV-2 with the cell surface receptor, angiotensin-converting enzyme 2 (ACE2), of the target cell, e.g. by direct blocking or by disrupting the pre-fusion conformation of the spike protein.
Blocking of the interaction between spike and ACE2 can be total or partial. For example, an antibody of the invention may reduce spike-ACE2 formation by ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100%. Blocking of spike-ACE2 formation can be measured by any suitable means known in the art, for example, by ELISA.
Most antibodies showing neutralisation also showed blocking of the interaction between the spike protein and ACE2. Furthermore, a number of non-neutralising antibodies are good ACE2 blockers.
In terms of binding kinetics, an antibody of the invention may have an affinity constant (KD) value for the spike protein of SARS-CoV-2 of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.4 nM, ≤0.3 nM, ≤0.2 nM or ≤0.1 nM.
The KD value can be measured by any suitable means known in the art, for example, by ELISA or Surface Plasmon Resonance (Biacore) at 25° C.
Binding affinity (KD) may be quantified by determining the dissociation constant (Kd) and association constant (Ka) for an antibody and its target. For example, the antibody may have an association constant (Ka) of ≥10000 M−1 s−1, ≥50000 M−1 s−1, ≥100000 M−1 s−1, ≥200000 M−1 s−1 or ≥500000 M−1 s−1, and/or a dissociation constant (Kd) of ≤0.001 s−1, ≤0.0005 s−1, ≤0.004 s−1, ≤0.003 s−1, ≤0.002 s−1 or ≤0.0001 s−1.
An antibody of the invention is preferably able to provide in vivo protection in coronavirus (e.g. SARS-CoV-2) infected animals. For example, administration of an antibody of the invention to coronavirus (e.g. SARS-CoV-2) infected animals 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.
Antibodies of the invention may have any combination of one or more of the above properties.
Antibodies of the invention may bind to the same epitope as, or compete for binding to SARS-CoV-2 spike protein with, any one of the antibodies described herein (i.e. in particular with antibodies with the heavy and light chain variable regions described above). Methods for identifying antibodies binding to the same epitope, or cross-competing with one another, are used in the Examples and discussed further below.
An antibody of the invention may or may not comprise an Fc domain.
The antibodies of the invention may be modified in the Fc region in order to improve their stability. Such modifications are known in the art. Modifications may improve the stability of the antibody during storage of the antibody. The in vivo half-life of the antibody may be improved by modifications of the Fc-region. For example, cysteine residue(s) can be introduced into the Fe region, thereby allowing interchain disulphide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922(1992)).
Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).
For example, an antibody of the invention may be modified to promote 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 interaction at low pH, such as in the endosome. The M252Y/S254T/T256E (YTE) mutation may be used to improve the half-life of an IgGI antibody.
The antibody may be modified to affect the interaction of the antibody with other receptors, such as FcγRI, FcγRIIA, FcγRIIB, FcγRIII, and FcaR. Such modifications may be used to affect the effector functions of the antibody.
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.
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 display the effector function or functions associated with a normal Fc region. An Fc region of an antibody of the invention does not bind to one or more Fc receptors.
In one embodiment, an antibody of the invention does not comprise a CH2 domain. In one embodiment, an antibody of the invention does not comprise a CH3 domain. In one embodiment, an antibody of the invention comprises additional CH2 and/or CH3 domains.
In one embodiment, an antibody of the invention does not bind Fc receptors. In one embodiment, an antibody of the invention does not bind complement. In an alternative embodiment, an antibody of the invention does not bind FcγR, but does bindcomplement.
In one embodiment, an antibody of the invention in general may comprise modifications that alter serum half-life of the antibody. Hence, in another embodiment, an antibody of the invention has Fc region modification(s) that alter the half-life of the antibody. Such modifications may be present as well as those that alter Fc functions. In one preferred embodiment, an antibody of the invention has modification(s) that alter the serum half-life of the antibody.
In one embodiment, an antibody of the invention may comprise a human constant region, for instance IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the IgG1 and IgG3 isotypes when the antibody molecule is intended for therapeutic uses where antibody effector functions are 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.
In one embodiment, the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either 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, either kappa or lambda.
The four human IgG isotypes bind the activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (Clq) with different affinities, yielding 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, an antibody of the invention does not bind to Fc receptors. In another embodiment of the invention, the antibody does bind to one or more type of Fc receptors.
In one embodiment the Fc region employed is mutated, in particular a mutation described herein. In one embodiment the Fc mutation is selected from the group comprising a mutation to remove or enhance binding of the Fc region to an Fc receptor, a mutation to increase or remove an effector function, a mutation to increase or decrease half-life of the antibody and a combination of the same. In one embodiment, where reference is made to the impact of a modification it may be demonstrated by comparison to the equivalent antibody but lacking the modification.
Some antibodies that selectively bind FcRn at pH 6.0, but not pH 7.4, exhibit a higher half-life in a variety of animal models. Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L (Hinton P R. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6) and M252Y/S254T/T256E+H433K/N434F (Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8), have been shown to increase the binding affinity to FcRn and the half-life of IgGI in vivo. Hence, modifications may be present at M252/S254/T256+H44/N434 that alter serum half-life and in particular M252Y/S254T/T256E+H433K/N434F may be present. In one embodiment, it is desired to increase half-life. In another embodiment, it may be actually desired to decrease serum half-life of the antibody and so modifications may be present that decrease serum half-life.
Numerous mutations have been made in the CH2 domain of human IgG1 and their effect on ADCC and CDC tested in vitro (Idusogie E E. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4):2571-5). Notably, alanine substitution at position 333 was reported to increase both ADCC and CDC. Hence, in one embodiment a modification at position 333 may be present, and in particular one that alters ability to recruit complement. Lazar et al. described a triple mutant (S239D/1332E/A330L) with a higher affinity for FcγRIIIa and a lower affinity for FcγRIIb resulting in enhanced ADCC (Lazar G A. et al., 2006). Hence, modifications at S239/I332/A330 may be present, particularly those that alter affinity for Fc receptors and in particular S239D/I332E/A330L. Engineered antibody Fc variants with enhanced effector function. PNAS 103(11): 4005-4010). The same mutations were used to generate an antibody with increased ADCC (Ryan M C. et al., 2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer Ther., 6: 3009-3018). Richards et al. studied a slightly different triple mutant (S239D/I332E/G236A) with improved FcγRIIIa affinity and FcγRIIa/FcγRIIb ratio that mediates enhanced phagocytosis of target cells by macrophages (Richards J O et al 2008. Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 7(8):2517-27). In one embodiment, S239D/I332E/G236A modifications may be therefore present.
In another embodiment, an antibody of the invention may have a modified hinge region and/or CH1 region. Alternatively, the isotype employed may be chosen as it has a particular hinge regions.
Public V-regions, also described as public V-genes herein, are the V regions of the germline heavy chain and light chain regions that are found in a large proportion of the antibody responses to SARS-CoV-2 found within the population. In this application, the V regions are specific responses to the Beta SARS-CoV-2 variant. That is to say, many individuals utilise the same v-regions from their germline v-region repertoire when generating an immune response to SARS-CoV-2 variants.
As used herein, an antibody “derived” from a specific v-region refers to antibodies that were generated by V(D)J recombination using that germline v-region sequence. For example, the 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. The nucleotide sequence encoding the antibody is unlikely to comprise a sequence identical to the IGHV3-53 germline sequence, nevertheless, the antibody is still derived from this v-region. An antibody of the invention typically comprises no more than non-silent mutations in the v-region, when compared to the germline sequence, such as no more than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 non-silent mutations. An antibody of the invention typically comprises no between 2-20 non-silent mutations in the v-region, when compared to the germline sequence, such as between 5-15, 6-13 and 7-12 non-silent mutations. Germline v-region sequences are well known in the art, and methods of identifying whether a certain region of an antibody is derived from a particular germline v-region sequence are also well known in the art.
In one embodiment, an antibody of the invention derives from a v-region selected from IGHV3-53, IGHV1-58, IGHV3-66, IGHV1-69, IGHV3-30, IGHV3-33, IGHV1-18, IGHV13-9 or IGHV4-31. The inventors found that the potent neutralising antibodies identified herein comprised relatively few mutations in the CDRs of these v-regions. Thus, in one embodiment, an antibody of the invention 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 having 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 the naturally occurring germline sequence. A silent mutation as defined herein is a change in the nucleotide sequence without a change in the amino acid sequence for which the nucleotide sequence encodes. A non-silent mutation is therefore a mutation that leads to a change in the amino acid sequence encoded by the nucleotide sequence.
The inventors have surprisingly found that the light chain variable region of two antibodies having the same heavy chain v-region may be exchanged to produce 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 may both comprise a heavy chain variable region derived from IGHV3-53. Preferably, both antibodies also comprise a light chain variable region derived from the same light chain v-region, although this is not essential because, for example, the light chain of antibody 222 may be matched with any heavy chain variable region derived from IGHV3-53 and lead to a potent neutralising antibody. As described above, the two antibodies may comprise a heavy chain variable region derived from IGHV3-53 and/or IGHV3-66.
In one embodiment, an antibody of the invention comprises the CDRs of an heavy chain variable domain of 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, such as 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 for IGHV1-58, 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 Omi 33 for IGHV3-33, antibodies antibody 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 SEQ ID NOs corresponding to the CDRs of each of these antibodies are shown in Tables 1, 2 and 3.
In one embodiment, an antibody of the invention comprises the heavy chain variable domain of 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, such as 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 for IGHV1-58, 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 Omi 33 for IGHV3-33, antibodies antibody 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 SEQ ID NOs corresponding to the CDRs of each of these antibodies are shown in Tables 1, 2 and 3.
In one embodiment, the invention provides a method of generating an antibody that binds specifically to the spike protein of SARS-CoV-2 (e.g. a SARS-CoV-2 strain of the Alpha, Beta, Gamma, Delta and/or Omicron lineages), the method comprising identifying two or more antibodies derived from the same light chain and/or heavy chain v-regions, replacing the light chain of a first antibody with the light chain of a second antibody, to thereby generate a mixed-chain antibody comprising the heavy chain of the first antibody and the light chain of the second antibody. In one embodiment, the method further comprises determining the affinity for and/or neutralisation of SARS-CoV-2 of the mixed-chain antibody. The method may further comprise comparing the affinity of the mixed-chain antibody with that of the first and/or second antibodies. The method may further comprise selecting a mixed chain antibody that has the same or greater affinity than the first and/or second antibodies. In some embodiments, the heavy chain v-region is IGHV 1-58 and/or the light chain v-region is IGLV Kappa 3-20.
In another embodiment, the invention provides an antibody that specifically binds to the Omicron variant of SARS-CoV-2, wherein the antibody has a v-region derived from IGHV1-69. It has been surprisingly discovered that antibody responses to infection with the Omicron variant of SARS-CoV-2 is biased towards antibodies with a heavy chain variable region derived from IGHV1-69. In one embodiment, wherein the antibody heavy chain is derived from IGHV1-69, the antibody of the invention comprises the CDRH1, CDRH2 and CDRH3 from Beta-49, Beta-50, Omi02, Omi24, Omi30, Omi31, Omi34 and Omi38.
The invention also pertains 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 fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein-coupling agents known in the art.
An antibody, of the invention may be conjugated to a molecule that modulates or alters serum half-life. An antibody, of the invention may bind to albumin, for example in order to modulate the serum half-life. In one embodiment, an antibody of the invention will also include a binding region specific for albumin. In another embodiment, an antibody of the invention may include a peptide linker which is an albumin binding peptide. Examples of albumin binding peptides are included in WO2015/197772 and WO2007/106120 the entirety of which are incorporated by reference.
The invention also provides one or more isolated polynucleotides (e.g. DNA) encoding the antibody of the invention. In one embodiment, the polynucleotide sequence is collectively present on more than one polynucleotide, but collectively together they are able to encode an antibody of the invention. For example, the polynucleotides may encode the heavy and/or light chain variable regions(s) of an antibody of the invention. The polynucleotides may encode the full heavy and/or light chain of an antibody of the invention. Typically, one polynucleotide would encode each of the heavy and light chains.
Polynucleotides which encode an antibody of the invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences. General methods by which the vectors may 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, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing. A polynucleotide of the invention may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the antibody of the invention in vivo. Hence, the invention also provides one or more expression cassettes encoding the one or more polynucleotides that encoding an antibody of the invention. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors). Hence, in one embodiment, the invention provides a vector encoding an antibody of the invention. In another embodiment, the invention provides vectors which collectively encode an antibody of the invention. The vectors may be cloning vectors or expression vectors. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention. The polynucleotides, expression cassettes or vectors of the invention are introduced into a host cell, e.g. by transfection. Hence, the invention also provides a host cell comprising the one or more polynucleotides, expression cassettes or vectors of the invention. The polynucleotides, expression cassettes or vectors of the invention may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the 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 bacteria cells. Particular examples of cells include mammalian HEK293, such as HEK293F,HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NSO and COS cells, or any other cell line used herein, such as the ones used in the Examples. Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation.
The invention also provides a process for the production of an antibody of the invention, comprising culturing a host cell containing one or more vectors of the invention under conditions suitable for the expression of the antibody from the one or more polynucleotides of the invention, and isolating the antibody from said culture.
The inventors found that certain Table 3 antibodies are particularly effective when used in combination, and certain combinations of Table 3, Table 2, and Table 1 antibodies, e.g. to minimise loss of activity due to SARS-CoV-2 variants, maximise therapeutic effects and/or increase diagnostic power. Useful combinations include the antibodies that do not cross-compete with one another and/or bind to non-overlapping epitopes.
Thus, the invention provides a combination of the antibodies of the invention, wherein each antibody is capable of binding to the spike protein of coronavirus SARS-CoV-2, wherein at least one antibody comprises at least three CDRs of any one of the 28 antibodies in Table 3.
A combination of the antibodies of the invention may be useful as a therapeutic cocktail. Hence, the invention also provides a pharmaceutical composition comprising a combination of the antibodies of the invention, as explained further below.
A combination of the antibodies of the invention may be useful for diagnosis. Hence, the invention also provides a diagnostic kit comprising a combination of the antibodies of the invention. Also provided herein are methods of diagnosing a disease or complication associated with coronavirus infections in a subject, as explained further below. A fully cross-neutralising antibody, e.g. Omi03, may be used as a reference to confirm the presence and/or amount of any variants of concern (VoC) SARS-CoV-2 in the sample. An antibody that binds to a limited number of VoCs may be used to confirm the presence and/or amount of that VoC in the sample. For example, if Omi03 exhibits binding to the sample but Omi24 does not exhibit binding to the sample of SARS-CoV-2, then the spike protein may be the spike protein of the Delta VoC. This may be determined by any method known to the skilled person, such as via an immunoassay, e.g. an ELISA or an immunochromatographic assay. Reduced binding may be determined by comparison and/or normalisation to the reference, and/or by comparison to positive/negative control samples or data.
The invention provides a pharmaceutical composition comprising an antibody of the invention. The composition may comprise a combination (such as two, three or four) of the antibodies of the invention. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.
The composition of the invention may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.
Suitable pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water and saline.
Other suitable pharmaceutically acceptable carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Therapeutic 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.
Pharmaceutical compositions of the invention may comprise additional therapeutic agents, for example an anti-viral agent. The anti-viral agent may bind to coronavirus and inhibit viral activity. Alternatively, the anti-viral agent may not bind directly to coronavirus but still affect viral activity/infectivity. The anti-viral agent could be a further anti-coronavirus antibody, which binds somewhere on SARS-CoV-2 other than the spike protein. Examples of an anti-viral agent useful with the invention include Remdesivir, Lopinavir, ritonavir, APN01, and Favilavir.
The additional therapeutic agent may be an anti-inflammatory agent, such as a corticosteroid (e.g. Dexamethasone) or a non-steroidal anti-inflammatory drug (e.g. Tocilizumab).
The additional therapeutic agent may be an anti-coronavirus vaccine. The pharmaceutical composition may be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally. Also within the scope of the invention are kits comprising antibodies or other compositions of the invention and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed herein.
The invention further relates to the use of the antibodies, the combinations of the antibodies and the pharmaceutical compositions, described herein, e.g. in a method for treatment of the human or animal body by therapy, or in a diagnostic method. The method of treatment may be therapeutic or prophylactic.
For example, the invention relates to methods of treating coronavirus (e.g. SARS-CoV-2) infections, a disease or complication associated therewith, e.g. COVID-19. The method may comprise administering a therapeutically effective amount of an antibody, a combination of antibodies, or a pharmaceutical composition of the invention. The method may further comprise identifying the presence of coronavirus, or fragments thereof, in a sample, e.g. SARS-CoV-2, from the subject. The invention also relates to an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for use in a method of treating coronavirus (e.g. SARS-CoV-2) infections, a disease or complication associated therewith, e.g. COVID-19.
The invention also relates to a method of formulating a composition for treating coronavirus (e.g. SARS-CoV-2) infections, a disease or complication associatedtherewith, e.g. COVID-19, wherein said method comprises mixing an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention with an acceptable carrier to prepare said composition.
The invention also relates to the use of an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for treating coronavirus (e.g. SARS-CoV-2) infections or a disease or complication associated therewith, e.g. COVID-19.
The invention also relates to the use of an antibody, a combination of antibodies, or a pharmaceutical composition according to the invention for the manufacture of a medicament for treating or preventing coronavirus (e.g. SARS-CoV-2) infections or a disease or complication associated therewith, e.g. COVID-19.
The invention also relates to preventing, treating or diagnosing coronavirus infection caused by any SARS-CoV-2 strain. The coronavirus infection may be caused by any SARS-CoV-2 strain.
The SARS-CoV-2 strain may 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 may be a member of lineage 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 may be a member of lineage A.23.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.258, B.1.526.2, B.1.616, B.1.617.1 (kappa), B.1.617.2 (delta), C36.3, C.37 (lambda), P.1 (gamma), B.1.1.529 (omicron), Omicron BA.1, Omicron BA.1.1, Omicron BA.2 and/or Omicron BA.3.
The SARS-CoV-2 strain may comprise one or more mutations, e.g. in the spike protein, relative to the hCoV-19/Wuhan/WIV04/2019 (WIV04) (GISAID accession no. EPI_ISL_402124). In other words, the SARS-CoV-2 strain may be a modified hCoV-19/Wuhan/WIV04/2019 (WIV04) strain comprising one or more modifications, e.g. in the spike protein.
The mutation may be the mutations (e.g. substitutions) observed in the Omicron strain of SARS-CoV-2.
Antibodies Omi02, Omi03, Omi12, Omi18, Omi28, Omi39 and Omi42 are particularly effective in neutralising the Omicron SARS-Cov-2 strain. Hence, the invention may relate to these antibodies for use in treating, prevent, treating or diagnosing coronavirus infection caused by a SARS-Cov-2 strain.
The methods and uses of the invention may comprise inhibiting the disease state (such as COVID-19), e.g. arresting its development; and/or relieving the disease state (such as COVID-19), e.g. causing regression of the disease state until a desired endpoint is reached.
The methods and uses of the invention may comprise the amelioration or the reduction of the severity, duration or frequency of a symptom of the disease state (such as COVID-19) (e.g. lessen the pain or discomfort), and such amelioration may or may not be directly affecting the disease. The symptoms or complications may be fever, headache, fatigue, loss of appetite, myalgia, diarrhoea, vomiting, abdominal pain, dehydration, respiratory tract infections, cytokine storm, acute respiratory distress syndrome (ARDS) sepsis, and/or organ failure (e.g. heart, kidneys, liver, GI, lungs).
The methods and uses of the invention may lead to a decrease in the viral load of coronavirus (e.g. SARS-CoV-2), e.g. by ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or 100% compared to pre-treatment. Methods of determining viral load are well known in the art, e.g. infection assays.
The methods and uses of the invention may comprise preventing the coronavirus infection from occurring in a subject (e.g. humans), in particular, when such subject is predisposed to complications associated with coronavirus infection.
The invention also relates to identifying subjects that have a coronavirus infection, such as by SARS-CoV-2. For example, the methods and uses of the invention may involve identifying the presence of coronavirus (e.g. SARS-CoV-2), or a protein or a fragment thereof, in a sample. The detection may be carried out in vitro or in vivo. In certain embodiments, the invention relates to population screening.
The invention relates to identifying any SARS-CoV-2 strain, as described herein. The invention may also relate to a method of identifying escape mutants of SARS-CoV-2, comprising contacting a sample with a combination of antibodies of the invention and identifying if each antibody binds to the virus. The term “escape mutants” refers to variants of SARS-CoV-2 comprising non-silent mutations that may affect the efficacy of existing treatments of SARS-CoV-2 infection. Typically, the non-silent mutations is on an epitope recognised by a prior art antibody and/or antibodies described herein that specifically binds to an epitope of SARS-CoV-2, e.g. on the spike protein of SARS-CoV-2. If the antibody does not bind to the target, it may indicate that the target comprises a mutation that may alter the efficacy of existing SARS-CoV-2 treatments.
The methods and uses of the invention may include contacting a sample with an antibody or a combination of the antibodies of the invention, and detecting the presence or absence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates that the subject is infected with SARS-CoV-2.
Methods of determining the presence of an antibody-antigen complex are known in the art. For example, in vitro detection techniques include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vivo techniques include introducing into a subject a labelled anti-analyte protein antibody. For example, the antibody can be labelled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. The detection techniques may provide a qualitative or a quantitative readout depending on the assay employed.
Typically, the invention relates to methods and uses for a human subject in need thereof. However, non-human animals such as rats, rabbits, sheep, pigs, cows, cats, or dogs is also contemplated. The subject may be at risk of exposure to coronavirus infection, such as a healthcare worker or a person who has come into contact with an infected individual. A subject may have visited or be planning to visit a country known or suspected of having a coronavirus outbreak. A subject may also be at greater risk, such as an immunocompromised individual, for example an individual receiving immunosuppressive therapy or an individual suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS). The subject may be asymptomatic or pre-symptomatic.
The subject may be early, middle or late phase of the disease.
The subject may be in hospital or in the community at first presentation, and/or later times in hospital.
The subject may be male or female.
In certain embodiments, the subject is typically male. The subject may not have been infected with coronavirus, such as SARS-CoV-2. The subject may have a predisposition to the more severe symptoms or complications associated with coronavirus infections. The method or use of the invention may comprise a step of identifying whether or not a patient is at risk of developing the more severe symptoms or complications associated with coronavirus.
In embodiments of the invention relating to prevention or treatment, the subject may or may not have been diagnosed to be infected with coronavirus, such as SARS-CoV-2.
The invention relates to analysing samples from subjects. The sample may be tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The sample may be blood and a fraction or component of blood including blood serum, blood plasma, or lymph. Typically, the sample is from a throat swab, nasal swab, or saliva.
The antibody-antigen complex detection assays may be performed in situ, in which case the sample is a tissue section (fixed and/or frozen) of the tissue obtained from biopsies or resections from a subject.
In the embodiments of the invention where the antibodies pharmaceutical compositions and combinations are administered, they may be administered subcutaneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially. Typically, the antibodies pharmaceutical compositions and combinations are administered intravenously or subcutaneously.
The dose of an antibody may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration. Antibodies may be administered at a dose of about 0.1 mg/kg body weight to a dose of about 100 mg/kg body weight, such as at a dose of about 5 mg/kg to about 10 mg/kg. Antibodies may also be administered at a dose of about 50 mg/kg, 10 mg/kg or about 5 mg/kg body weight.
A combination of the invention may for example be administered at a dose of about 5 mg/kg to about 10 mg/kg for each antibody, or at a dose of about 10 mg/kg or about 5 mg/kg for each antibody. Alternatively, a combination may be administered at a dose of about 5 mg/kg total (e.g. a dose of 1.67 mg/kg of each antibody in a three antibody combination).
The antibody or combination of antibodies of the invention may be administered in a multiple dosage regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time.
As discussed above, the antibodies of the invention are typically used in a single pharmaceutical composition/combination (co-formulated). However, the invention also generally includes the combined use of antibodies of the invention in separate preparations/compositions. The invention also includes combined use of the antibodies with additional therapeutic agents, as described above.
Combined administration of the two or more agents and/or antibodies may be achieved in a number of different ways. In one embodiment, all the components may be administered together in a single composition. In another embodiment, each component may be administered separately as part of a combined therapy.
For example, the antibody of the invention may be administered before, after or concurrently with another antibody, or binding fragment thereof, of the invention. The particularly useful combinations are described above for example.
For example, the antibody of the invention may be administered before, after or concurrently with an anti-viral agent or an anti-inflammatory agent.
In embodiments where the invention relates to detecting the presence of coronavirus, e.g. SARS-CoV-2, or a protein or a fragment thereof, in a sample, the antibody contains a detectable label. Methods of attaching a label to an antibody are known in the art, e.g. by direct labelling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody. Alternatively, the antibody may be indirect labelled, e.g. by reactivity with another reagent that is directly labelled. Examples of indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently-labelled streptavidin.
The detection may further comprise: (i) an agent known to be useful for detecting the presence of coronavirus, e.g. SARS-CoV-2, or a protein or a fragment thereof, e.g. an antibody against other epitopes of the spike protein, or other proteins of the coronavirus, such as an anti-nucleocapsid antibody; and/or (ii) an agent known to not be capable of detecting the presence of coronavirus, e.g. SARS-CoV-2, or a fragment thereof, i.e. providing a negative control.
In certain embodiments, the antibody is modified to have increased stability. Suitable modifications are explained above.
The invention also encompasses kits for detecting the presence of coronavirus, e.g. SARS-CoV-2, in a sample. For example, the kit may comprise: a labelled antibody or a combination of labelled antibodies of the invention; means for determining the amount of coronavirus, e.g. SARS-CoV-2, in a sample; and means for comparing the amount of coronavirus, e.g. SARS-CoV-2, in the sample with a standard. The labelled antibody or the combination of labelled antibodies can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect coronavirus, e.g. SARS-CoV-2, in a sample. The kit may further comprise other agents known to be useful for detecting the presence of coronavirus, as discussed above.
For example, the antibodies or combinations of antibodies of the invention are used in a lateral flow test. Typically, the lateral flow test kit is a hand-held device with an absorbent pad, which based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. The test runs the liquid sample along the surface of the pad with reactive molecules that show a visual positive or negative result. The test may further comprise using other agents known to be useful for detecting the presence of coronavirus, e.g. SARS-CoV-2, or a fragment thereof, as discussed above, such as anti-an anti-nucleocapsid antibody.
It is to be understood that different applications of the disclosed antibodies combinations, or pharmaceutical compositions of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” includes two or more “antibodies”.
Furthermore, when referring to “≥x” herein, this means equal to or greater than x. When referred to “≤x” herein, this means less than or equal to x.
For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions in the reference sequence×100). Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished 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), the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those 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, the gaps or missing positions should be considered to be non-identical positions. The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The CDRs of the heavy chain (CDRH) and 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 M P, 1997, J, Immunol. Today, 18, 509). This numbering system is used in the present specification except where otherwise indicated.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
The following examples illustrate the invention.
The antibodies in Table 1 relate to a set of mAbs generated against early pandemic strain of SARS-CoV-2. The antibodies in Table 2 relate to a set of mAbs generated against the Beta strain of SARS-CoV-2.
Further details of these antibodies may be found in international application no. PCT/GB2022/050306 & PCT/GB2022/050307. Further information on the generation and properties of these antibodies may be found in the following articles: Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptorbinding 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.
Omicron BA.2 was first reported from South Africa on the 17 Nov. 2021, at a similar time that Omicron BA.1 was reported. BA.2 has been increasing relative to BA.1 in a number of countries such as Denmark, India and the UK and now accounts for the majority of Omicron infections in Denmark and evidence is accruing that BA.2 is more transmissible than BA. 1, but there is no evidence for increased disease severity.
BA.2 is related to BA.1 sharing 21 amino acid substitutions spread throughout in S, however there are a number of differences BA.1, has an additional 6 amino acid deletions, 3 insertions and 9 substitutions compared to BA.2 and BA.2 has an additional 3 deletions and 7 substitutions compared to BA.1. In the RBD, BA.1 contains unique mutations S371L, G446S and G496S and in some isolates R346K (BA.1.1), while BA.2 carries S371F, T376A, D405N and R408S. All of these residues have the potential to differentially affect antibody binding and could modulate neutralization, particularly BA.1 G446S, G496S, BA.2 D405N, R408S which lie at the edge of the ACE2 binding footprint and for BA.1.1 the R346K change lies close to the N343 glycan and could modulate binding of potent antibodies to this region. BA.3 contains no unique mutations relative to BA.1 and BA.2 and appears to be a fusion of the two, being BA.1 like at the N terminus and switching to become BA.2 like at the C-terminus from the mutation G496S.
In early April 2022 two new Omicron lineages were reported from Gauteng in South Africa and designated BA.4 and BA.5. The BA.4 and BA.5 S sequences are identical, and closely related to BA.2. Sequence diversity in Omicron S is shown in
The 2 additional mutations in the RBD are of most concern in terms of antibody escape: L452R is a chemically radical change and is one of the pair of changes in Delta RBD (the other, T478K, is already found in the Omicron lineage). Mutation F486L was found in sequences of SARS-CoV-2 isolated from Mink early in the pandemic and is also a site of escape mutations to several mAbs (Gobeil et al., 2021, “Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity”. Science 373, 6555). The change F486V in BA.4/5 is also a reduction in the bulk of the hydrophobic side-chain as in F486L, but more significant. Both residues 452 and 486 lie close to the edge of the ACE2 interaction surface (
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, 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, 1210V and G257S) and four in the RBD: D339H, G446S, N460K and R493Q (
N460K is a novel mutation not seen in previous VoC or Omicron sublineages, but it was found after in vitro (yeast display) evolution in RBD-62 which has an ultra-high ACE2 affinity (KD=16-18 pM) (Dejnirattisai et al., 2022; Zahradnik et al., 2021 “SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution”. Nat Microbiol 6, 1188-1198). Indeed N460K led to substantial increase in affinity for ACE2, second only to the effect of N501Y (Zahradnik et al., 2021). Furthermore, in silico analysis predicts that N460K may affect the binding of certain antibodies belonging to the IGHV3-53 family (e.g. Omi-3) which have been shown to be able to potently neutralise all VoC (Nutalai et al., 2022).
Using neutralization assays, Delta infection in isolation was show to provide no protection (no neutralization) against BA.2.75. The mutations in BA.2.75 lead to a reduction in neutralization titres of vaccine serum compared to BA.2. Individual BA.2.75 mutations can cause greater reduction in neutralization titres compared to the full BA.2.75 S sequence, but these are balanced by the R393Q reversion mutation, which may have been selected to increase affinity to ACE2 and increase the transmissibility of BA.2.75. It seems inevitable that further evolution of the Omicron lineage will occur and there are likely many possible trade-offs between antibody escape and ACE2 affinity, that can and will be made, leading to successive waves of infection.
A number of lineages are growing rapidly from within both the BA.2 and BA.5 branches. Most striking, 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 examples from the BA.4/5 branches (which contain 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); from the BA.2.75 branch (which contains G339H, G446S, N460K and the reversion R493Q), BA.2.75.2 (R346T and F486S and BA.2.75 mutations), BN.1 (aka BA.2.75.5.1 with R346T, K356T, F490S and BA.2.75 mutations), BM.1.1.1 (aka BA.2.75.3.1.1.1 with R346T, F486S, F490S and BA.2.75 mutations). There are also examples of several other second generation BA.2 variant lines such as BJ.1 (aka BA.2.10.1.1; G339H, R346T, L368I, V445P, G446S, V483A and F490V), BA.2.10.4 (G446S, F486P, S494P and the R493Q reversion), BS.1 (aka BA.2.3.2.1; R346T, L452R, N460K, G476S and the Q493R reversion), BA.2.3.20 (K444R, N450D, L452M, N460K, E484R and the Q493R reversion), and finally a BJ.1×BM.1.1.1 (aka BA.2.75.3.1.1.1) recombinant, XBB (which relative to BA.2 contains R346T, L368I, V445P, G446S, N460K, F486S, F490S and the Q493R reversion).
Outside the RBD the degree of convergent evolution is lesser but still present. Many of the second-generation BA.2 variant lineages contain deletions or mutations in the NTD, often similar to those seen in the VoCs, for example A˜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).
Five volunteers who had recovered from sequence confirmed Omicron infection were recruited and sampled them 10-14 days following symptom onset; all volunteers had received 2 doses of the Pfizer BioNtech vaccine before being infected with Omicron. First, neutralization assays were performed against Omicron BA.1 and Victoria (an early pandemic SARS-CoV-2 isolate containing only a single amino acid substitution in S NTD (S247R) compared to the sequence of the Wuhan stain used in all current vaccines). In all cases the focus reduction neutralization 50% titre (FRNT50) to Omicron was above 100, but at this early time point the titres were considerably below the titres to Victoria (
B cells from the five donors were stained with full length BA.1 trimer and single cells sorted by FACS (
All mAbs cross-reacted between WT and BA.1 S by ELISA, suggesting that they could have been generated from memory B cells induced by vaccination. In contrast to a previous panel of monoclonal antibodies were produced from naïve cases infected early during the pandemic (Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184.8 (2021): 2183-2200), a higher proportion the omicron-specific mAbs were found to react to the RBD (56%) when compared to the early pandemic mAbs (21%, p<0.0001) (
Neutralization assays were performed on all ELISA positive mAb and those showing the highest activity were chosen for further study. The most potent 28 mAbs were selected for full characterization all of which showed BA.1 FRNT50 titres <100 ng/ml. 27/28 bound the RBD (one, Omi-41 bound the NTD) and none cross-reacted with SARS-CoV-1 S protein by ELISA.
Examination of gene usage (
Roughly one half of the gene families observed in the potent early pandemic antibodies (Table 1) are also represented in the Omicron set (
Compared to the early pandemic set of antibodies we found higher levels of somatic mutation in both heavy and light chains compared to the early pandemic set of mAb Omicron (mean of 9.00, 6.00 and early pandemic 4.55, 4.25 for VH and VL respectively). These results would be consistent with the evolution of increased Omicron affinity via somatic mutation of vaccine induced memory B cells.
Neutralization assays were performed against Victoria and all variants of concern Alpha, Beta, Gamma, Delta and Omicron BA.1, for the panel of 28 potent mAbs (
The data provided in Tables 13, 14 and 16 include some IC50 data obtained using pseudoviral constructs. The data in Table 18 consists of IC50 results obtained exclusively from authentic virus constructs.
17/28 antibodies are cross-reactive against all VoC with <10-fold difference in FRNT50 titres between all viruses. Omi-06, 24, 30, 31, 34 and 41 show reduced or absent activity against Delta, with 3/6 of these belonging to the VH1-69 family, and may have an epitope impinging on the L452R Delta mutation (Delta shares T478K with BA. 1). Antibodies Omi-09 and 32 perform poorly on Beta and Gamma and may be sensitive to E484K found in Beta and Gamma, but may tolerate the E484A change in Omicron (Omicron shares N501Y and K417N with Beta whilst Gamma is N501Y, K417T). Finally, although 129 anti-NTD mAbs were isolated only one of these, Omi-41, showed FRNT50 titres <100 ng/ml, Omi-41 showed neutralizing activity against Victoria, Alpha, Beta and Gamma but no activity against Delta, presumably resulting from the unique spectrum of NTD changes found in Delta.
Neutralization of BA.1 compared to BA.1.1, BA.2 and BA.3 Lentiviral based reporters were constructed pseudotyped with the S gene sequences for Victoria, BA.1, BA.1.1, BA.2 and BA.3. Neutralization assays against the Omicron mAb are shown in
Pseudoviral neutralization curves for panels of mAb isolated from early pandemic cases together with mAb isolated from Beta cases is shown in
Finally, neutralization of Victoria, BA.1, BA.1.1, BA.2 and BA.3 strains was tested using mAbs being developed for clinical use where a number of differences were found (
In summary, the neutralization of most Omicron monoclonal antibodies are not affected by the differences between BA.1, BA.1.1, BA.2 or BA.3 mutations. Some monoclonal antibodies do however show differences, in particular Known Antibody A (REGN 10987) and Known antibody C (AZD1061) which neutralize BA.2 more easily than BA.1 and Known Antibody K (S309) which shows reduced neutralization of BA.2 and this may encourage sub-lineage typing before use. The structural explanations for the differences between BA.1, BA.1.1, BA.2 and BA.3 neutralization will be discussed below.
To determine whether the differences in transmissibility between BA.1 and BA.2 may be due to differential neutralization and also to determine whether there was a possibility that BA.2 could escape the BA.1 antibody response, neutralization assays were performed using sera from a variety of sources. First, neutralization assays were performed on Victoria, BA.1, BA.1.1, BA.2 and BA.3 using sera collected from vaccinees receiving the Oxford/AstraZeneca AZD1222 (n=41) or Pfizer/BioNtech BNT162b2 (n=20) vaccines (
For AZD1222 samples were taken 4 weeks after the second and third doses of vaccine. Following the third dose of AZD1222 there were small but significant differences between pseudoviral neutralization with reductions of the titres 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 following the second dose of vaccine, before the third dose and 4 weeks after the third dose. Following the third vaccine dose, titres against BA.1, BA.2 and BA.1.1 were similar with non-significant differences between them. Next, the neutralization profile of serum collected from cases infected with Omicron were determined. Early samples (n=12) were taken <14 days from symptom onset (median 13 days), later samples (n=17) were taken >21 days following symptom onset (median 38 days). All cases had received at least 2 doses of vaccine and a number of the late convalescent cases received a third dose of vaccine following Omicron infection. Neutralization against Victoria, Alpha, Beta, Gamma, Delta and Omicron was tested using live virus neutralization assays (
Neutralization of Victoria, BA.1, BA.1.1, BA.2 and BA.3 was assayed by pseudoviral neutralization. BA.1 neutralization titres were higher at later time points. However, all of the sera were obtained from BA.1 infected cases and there were small but significant reductions in the neutralization titres of BA.2 vs BA.1 (1.7 and 1.5-fold p=0.0034 and 0.0067 at <14 and >21 days respectively), the titres of BA.1.1 vs BA.1 were not significantly reduced while at >21 days the titre against BA.3 vs BA.1 was reduced 1.7-fold (p=0.0012) (
In summary, following three doses of vaccine, particularly BNT162b2, good neutralizing titres of antibody against Omicron BA.1 BA.1.1, BA.2 and BA.3 are induced, with only minor differences between the titre 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. Following break through Omicron infection, in previously vaccinated individuals, there is boosting of a broad antibody response to variants of concern and the generation of strong responses to Omicron. Since there are only small differences in the neutralization between BA.1 and BA.2, BA.2 superinfection of BA.1 exposed and vaccinated cases is unlikely, at least in the short term.
Neutralization of BA.4/5 was also assessed in comparison to Omicron sub-lineages BA.1, BA.1.1, BA.2, BA.3 and the early pandemic Victoria strain. BA.4/5 was shown to have a more extreme antibody escape phenotype than BA.1 and BA.2, and serum from triple vaccinated donors had ˜2-3-fold reduction in neutralization titres compared to the neutralization of BA.1 and BA.2. Additionally, serum from breakthrough BA.1 infections in vaccinees showed ˜2-3-fold reduction in neutralization titres to BA.4/5 compared to BA.1 and BA.2. This suggests that currently approved vaccines and mAbs may be less effective at preventing BA.4/5 transmission. New monoclonals and combinations may therefore be needed to plug the gap to protect the extremely vulnerable and those unable to mount adequate vaccine responses.
A panel of pseudotyped lentiviruses (Di Genova et al., 2020, “Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes”. Figshare preprint.) expressing the S gene from the Omicron sub-lineages BA.1, BA.1.1, BA.2, BA.3 and BA.4/5 was constructed, together with early pandemic Wuhan related strain, Victoria, used as a control.
Neutralization assays were performed using serum obtained 28 days following a 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) (
Neutralization of BA.4/5 by Serum from Breakthrough BA.1 Infection
At the onset of the Omicron outbreak, vaccinated volunteers who had suffered breakthrough Omicron infections were recruited. Samples were first taken 14 days from symptom onset (median 13 days), while late samples were taken ≥21 days from symptom onset (median 38 days) n=16. Pseudoviral neutralization assays were performed against the panel of pseudoviruses representing variants of concern and the Omicron Sub-lineages (
BA.1 infection following vaccination leads to a broad neutralizing response, with high titres to all the VoC, which is boosted at later time points (Nutalai et al. 2022, “Potent cross-reactive antibodies following Omicron breakthrough in vaccines”. Cell (in press)). Neutralization titres against BA.4 were significantly less than BA.1 and BA.2, at the early time point BA.4/5 titres were reduced 1.9-fold (p=0.0001) and 1.5-fold (p=0.0015) compared to BA.1 and BA.2 respectively. At the later point BA.4/5 titres were reduced 3.4-fold (p=0.0001) and 2-fold (p=0.0017) compared to BA.1 and BA.2 respectively.
Thus, BA.4/5 shows a degree of immune escape from the vaccine/BA.1 response when compared with BA.1 and BA.2. These samples were all taken reasonably close to the time of infection meaning that further waning in the intervening months may render individuals susceptible to reinfection with BA.4/5.
Escape from Monoclonal Antibodies by BA.4/5
Sensitivity to L452R: It has previously been reported that Omi-24, 30, 31, 34 and 41 show complete knock out of neutralizing activity against Delta, with Omi-06 showing severe knock-down of activity (Nutalai et al., 2022). Since BA.1 and BA.2 harbour only one (T478K) of the 2 Delta RBD mutations, whilst BA.4/5 also harbour L452R, it is expected that all five of these L452 directed mAbs to be knocked out on BA.4/5. This is indeed observed (
To confirm that the neutralization effects observed are directly attributable to alterations in RBD interactions, binding analyses of selected antibodies to BA.4/5 and BA.2 RBDs by surface plasmon resonance (SPR) were also performed (
Since detailed information on the interaction of several Omicron responsive antibodies with the RBD is available, the BA.4/5 RBD mutations were modelled in the context of known structures for Omicron Fabs complexed with BA.1 or Delta RBDs (Dejnirattisai et al., 2022, “SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses”. Cell 185, 467-484 e415; Nutalai et al., 2022), (
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 RBDs suggests that this is mainly achieved by a 5-fold increase in the on-rate of binding (
Sensitivity to F486V: Extending the logic used to understand Delta sensitivity, the remaining antibodies affected by BA.4/5>BA.2, but which retain activity against Delta, are likely sensitive to the F486V change, namely Omi-02, 09, 12, 23, 25, 26, 29. The binding sensitivity was confirmed by SPR analysis of Omi-12 (
A panel of antibodies that have been developed for therapeutic/prophylactic use was tested against BA.4/5 (
These effects can be rationalized by reference to the way the antibodies interact with the RBD, for instance in the case of AZD8895 (an IGHV1-58 genotype mAb,
A panel of pseudotyped lentiviruses was constructed as above (Di Genova et al., 2020) expressing the S gene from the Omicron sub-lineages BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, BA.2.75, together with Victoria, an early pandemic Wuhan related strain, used as a control. D339H, G446S, N460K and R493Q were also included as single mutations on the BA.2 background. Neutralization assays were performed using serum obtained 28 days following a 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 of Pfizer-BioNtech vaccine BNT162b2 (n=22) (Cele et al., 2021; “Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization”. Nature 602, 654-666e) (
Neutralization of BA.2.75 by Serum from Vaccine Breakthrough BA.1 or BA.2 Infections
Breakthrough BA.1 serum samples were taken from vaccinated volunteers ≥28 days from symptom onset (median 38 days; n=16). Pseudoviral neutralization assays were performed against the panel of pseudoviruses described above (
Breakthrough BA.2 serum samples were taken from vaccinated volunteers 12 days from symptom onset (median 29 days; n=23). Pseudoviral neutralization assays were performed against the panel of pseudoviruses Victoria, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5 and BA.2.75 (
Individual BA.2.75 Mutation have Differential Effects on Neutralization
To understand the effects of the individual mutations in the BA.2.75 RBD, these mutations were introduced individually into the pseudovirus BA.2 background and their neutralization was assayed using triple vaccinated Pfizer BNT162b2 serum (
Escape from Monoclonal Antibodies by BA.2.75
To dissect how BA.2.75 might affect neutralising antibody activity, pseudoviral assays were used to test a recently reported panel of potent human mAbs generated from cases of Omicron breakthrough infection (BA.1 IC50 titres <0.1 μg/ml) (Nutalai et al., 2022.) (
Like BA.2 and BA.4/5, BA.2.75 is not neutralised by the anti-NTD mAb Omi-41, which only interacts with the NTD of BA.1, BA.1.1 and BA.3.
The Omi mAbs were also tested against the pseudoviruses encoding single point mutations in the BA.2 RBD described above (
Interestingly, BA.2.75 is more sensitive to Omi-32 (IGHV-3-33) than BA.2, with an 8-fold increase in neutralisation titre. The enhancement in activity by Omi-32 is likely due to a stronger interaction of the antibody with the RBD through the G446S mutation (
To confirm that the change in neutralising activities observed are associated with alterations in RBD interaction, binding analyses of selected antibodies to BA.2.75 and BA.2 RBDs were performed by surface plasmon resonance (SPR) (
Escape from Commercial Monoclonals Against BA.2.75
The sensitivity of a panel of mAbs that have been developed as therapeutics against BA.2.75 (
Escape from Monoclonal Antibodies by BA.2, BA.4 and BA.5 Sublineages
Attrition of mAb activity was also observed with the new BA.2, BA.4 and BA.5 sublineages (including BA.4.6, BA.2.75, BA.2.75.2, BA.2.3.20, BJ.1, BQ.1, BQ.1.1, XBB, XBB.1 and XBB.1.5) (
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.
The receptor binding capacity of the BA.2 subvariants BA.2.11, BA.2.12 and BA.2.13 was also evaluated. A high-resolution crystal structure of BA.2.12.1 RBD was generated, showing differential sensitivity of 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.
Considering the physico-chemical properties of the side chain of residue 452, BA.2.13 would be expected to be a relatively modest change; L to M will increase the size of the side chain but it remains hydrophobic. L to Q in BA.2.12.1 introduces some polar character, whilst BA.2.11 is the most radical with L to R introducing a large basic amino acid.
To evaluate the susceptibility of the BA.2 subvariants to neutralisation by immune sera, neutralization assays were performed on pseudotyped lentiviruses expressing the Spike gene of BA.2.11, BA.2.12 and BA.2.13, using a series of serum samples.
Firstly, the neutralisation profile with sera collected 4 weeks following a third dose of the Oxford-AstraZeneca vaccine AZD1222 (n=41) or Pfizer-BioNtech vaccine BNT162b2 (n=18) was observed. No significant loss in neutralisation titre was seen compared to BA.2. In fact, BA.2.13 showed a significant increase (1.6-fold, p<0.0001) for AZD1222 vaccinees (
Neutralisation of BA.2 Subvariants BA.2.11, BA.2.12 and BA.2.13 by Serum from Vaccine Breakthrough BA.1 or BA.2 Infections
Next, the neutralisation profile for serum samples collected from vaccinees infected with BA.1 were examined. Samples (n=14) were taken ≥28 days following symptom onset (median 38 days); all convalescent individuals had received at least 2 doses of vaccine, 3 of them received a third dose of vaccine following Omicron infection. There were significant reductions in neutralisation titre for all three variants compared to BA.2, with the greatest decrease for 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) (
To further elucidate the differential responses between BA.2 and its subvariants, pseudoviral assays were performed on a panel of potent human monoclonal antibodies (mAbs) generated from cases of BA.1 breakthrough infection (Nutalai, et al., 2022). (
The spike mutations in the BA.2.11, BA.2.12 and BA.2.13 variants could render it slightly more transmissible than BA.2. However, compared to BA.2, they do not appear to have acquired greater humoral immune escape in healthy vaccinees who have received three doses of the Oxford-AstraZeneca or Pfizer-BioNtech BNT162b2 vaccine. This result differs from that of vaccinees who have received the triple-dose CoronaVac vaccine, for whom significant reductions in neutralisation titres were observed (Cao, Y., et al., BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature, 2022). Nevertheless, significant reductions in neutralisation titres were seen in vaccinees who had experienced BA.1 breakthrough infections, no matter which type of vaccine was received, perhaps partly due to partial or complete knock-out of neutralising activity of antibodies belonging to the IGVH1-69 family, many of which are sensitive the mutation at leucine 452 of the Spike RBD. This suggests that the continuously evolving Omicron sublineages are able to gain evasion from the humoral immune responses mounted by BA.1, thus implying that BA.1 Spike or RBD might not be a substantially better immunogen than that of the ancestral Wuhan strain for the development of the next-generation SARS-CoV-2 vaccine.
The crystal structure of BA.2.12.1 RBD was determined at 2.38 Å as a ternary complex with a neutralizing Fab and nanobody (
Neutralisation of BA.2.75.2 by mAbs Made Following BA.1 Infection
Neutralisation of BA.2.75.2 by a panel of mAbs made following BA.1 infection (Nutalai et al., 2022) was investgated. Attrition of mAb activity was observed against BA.2.75.2 (Table 32a). Activity of all 9 IGVH3-53/66 mAbs was reduced >100-fold with complete knock out of activity in 4/9 by BA.2.75.2. Only a single mAb, Omi-42 was unaffected by all variants showing neutralization of the BA.4+14 mutations described with IC50 of 11 ng/ml. Omi-42 is unusual as it binds at the back of the left shoulder of the RBD (Nutalai et al., 2022) in a region that has not yet been targeted for mutation, perhaps because of the relative rarity of antibodies binding in this region.
A panel of mAb that have been developed for clinical use was also tested (Dong et al., 2021; Sun and Ho, 2020; Weinreich et al., 2021; Yuan et al., 2022). Many of these were severely impacted by a number of variants. Activity of all mAbs apart from S309 was knocked out by one or more variants including Ly-CoV1404 (Westendorf et al., 2022) (see Table 32b).
Neutralization on serum collected 28 days following a third dose of Pfizer BNT162b2 vaccine (Polack et al., 2020) and in cases infected with BA.1, BA.2 of BA.4/5 the characteristics of these subjects are described in the methods.
Using serum obtained 28 days following BNT162b, infection titres to BA.2.75.2 showed large reductions compared to BA.2 and BA.4, and were the lowest of all variants tested compared to the ancestral strain Victoria. The reduction in titres to BA.2.75.2 were in contrast to BA.2.75 which showed only a modest reduction compared to BA.2. There were also large reductions in titres to BA.2.3.20.
Using serum obtained following BA.1, BA.2 and BA.4/5 infection, there were similar large reductions in the neutralization titres of BA.2.75.2 and BA.2.3.20 compared to BA.4/5. Neutralization of the BA.4+14 RBD mutations described above were also reduced compared to BA.2 and BA.4/5 but not a great deal more than BA.2.75.2 indicating a dominant effect of mutations in BA.2.75.2
Neutralisation of BA4.6 by Serum from Vaccine Breakthrough BA.1 or BA.2 Infections
Here, we study the neutralisation profile of BA.4.6 using: Pfizer-BioNtech vaccine serum, BA.1, BA.2 and BA.4/5 vaccine breakthrough immune serum, as well as panels of monoclonal antibodies. Remarkably, we show further antibody evasion of BA.4.6, providing guidance for vaccine design and the use of therapeutic monoclonals.
To evaluate the antibody evasion capacity of BA.4.6, we constructed a panel of pseudotyped lentiviruses (Di Genova, C., et al., Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes. figshare, 2020.) expressing the S gene from BA.4.6 and other SARS-CoV-2 variants together with early pandemic Wuhan related strain, Victoria, used as a control. Firstly, the neutralisation profile was examined with sera collected 4 weeks following a third dose of the Pfizer-BioNtech vaccine BNT162b2 (n=22). Compared to BA.4/5, neutralisation titres against BA.4.6 were reduced 2-fold (p<0.0001) for BNT162b2 sera (
The neutralisation profile for serum samples collected from vaccinees infected with BA.1 were assated. Samples (n=16) were taken ≥28 days following symptom onset. BA.2 samples (n=23) were taken ≥12 days following symptom onset or BA.4/5. Samples (n=11; all but one vaccinated) were taken >23 days following symptom onset (
To further characterise the antigenic escape properties of BA.4.6, pseudoviral assays were performed on a panel of potent human mAbs generated from BA.1 breakthrough convalescents (Nutalai et al., 2022) (
Neutralisation of BA.4.6, by mAbs in Clinical Use
Finally, neutralisation activities of a number of mAbs in clinical use was evaluated (
In summary, BA.4.6 showed further reduction in neutralisation by serum from triple dose Pfizer vaccinees, as well as from BA.1 and BA.2 vaccine breakthrough convalescents compared to BA.4/5. Notably, BA.4.6 does not seem to more resistant to neutralisation by serum from BA.4/5 breakthrough infection compared to other variants. This altogether suggests that there is a strong likelihood of infection or breakthrough infection by BA.4.6 unless one has been triply vaccinated and recovered from BA.4/5 infections, which seems to provide some protection against BA.4.6.
As of September 2022, bivalent booster vaccination, combining the ancestral strain with Omicron BA.1 is being rolled out in the UK, and has been recently authorised by FDA. It remains to be seen how effective these bivalent boosters are at preventing BA.4.6 infection. Finally, BA.4.6 has further impaired the activity of Evusheld which remained active against BA.4/5; as a result, now only LY-CoV1404/bebtelovimab retains potency against all circulating SARS-CoV-2 variants.
Both Omi-3 (a representative of the IGVH3-53 gene family) and AZD8895 (IGVH1-58) make contacts with F486. Whilst the F486V mutation has little effect on Omi-3 (
The effects on antibodies with broadly similar epitopes can vary dramatically, and this is equally true for antibodies which have 452 or 486 central to their binding footprint. Thus Omi-31 (IGVH1-69) and Omi-32 (IGVH3-33), both bind in front of the right shoulder with their CDR-H3 positioned close to 452, whilst the activity of Omi-31 is abolished by L452R (as detailed above), Omi-32 is markedly enhanced (
A matrix of pairwise BLI measurements were used to map the potent RBD binding Omicron mAbs and several pre-pandemic mAbs of known binding position.
The method yielded a consistent prediction. The mAbs segregate into a restricted set of epitopes, which appear to be subset of the epitopes observed for the early pandemic virus, and are quite distinct from the focus seen for Beta. Essentially the antibodies cluster in two regions, one which includes the VH3-53 and VH3-66 type antibodies is towards the back of the neck/left shoulder, extending up to the top of the left shoulder, whilst the other is on the front of the neck right shoulder region, spilling towards the S309 known antibody binding site. This region is occupied by the VH1-69 family antibodies, with the exception of Omi-2 which is sited within the other cluster. mAb Omi-09 which shows reduced neutralization of Beta and Gamma positions close to residue 484 which is mutated from Glu to Lys in Beta/Gamma and Ala in Omicron. VH1-69 mAb Omi-24, 30, 31 and 34, which show reduced neutralization of Delta are placed close to residue 452 which is mutated from Leu to Arg in Delta.
Structural analyses of selected potent Omicron mAbs were performed. Crystal structures were determined for complexes of Omicron BA.1 RBD with 3 different Fabs: Omi-3, 9 and 12. The complex of Omi-12 was at low resolution (5.5 Å) and so the structure of the Fab alone was determined at high resolution and rigid-body fitted to obtain the complex structure.
Omi-3 belongs to the VH3-53 gene family and demonstrates how this gene family can be adapted to be broadly neutralising against all major SARS-CoV-2 variants (but, like all the potent Omicron antibodies it does not bind SARS-CoV-1 RBD). A fundamental problem for these antibodies is that most VoC harbour mutation N501Y, which introduces a steric clash with the LC CDR1 (L1) capable of abrogating the binding of the large majority of VH3-53 containing antibodies. However, two mechanisms for displacing L1 to avoid this clash have been previously reported (Dejnirattisai et al., 2021b; Liu et al., 2021b). In mAb-222, isolated from individuals infected with early pandemic strains, a proline is inserted at residue 30 which can pack against Tyr-501 without clashes (Dejnirattisai et al., 2021b), allowing it to effectively neutralize Alpha, Beta and Gamma variants. Beta-27 uses an alternative mechanism, lengthening the HC CDR3 (H3) loop to 11 residues from the usual 9, displacing L1 to produce enough space to allow 501Y to be stabilised by main chain interactions conferring similar cross-reactivity (Liu et al., 2021b).
Omi-3 uses the same mechanism as Beta-27 for accommodating the N501Y mutation, although the Omi-3 H3 is one residue longer again. Other VH3-53 Omicron antibodies (Omi-18 and Omi-29) have H3s very similar to Beta-27 and presumably usethe same mechanism. This L1 configuration is also compatible with the Y505H mutation in Omicron. However, neither 222 nor Beta-27 can effectively neutralize Omicron and this may be due to specific features of the H3 loop which makes close contact with the Q493R Omicron mutation.
Omi-9 is a one of three VH3-30 mAbs and binds across the left shoulder of the RBD. Omi-9 shows relatively weak neutralization of Beta and Gamma (
Omi-12 belongs to the VH1-58 gene family (it is the only member of this family amongst the 28 potent Omicron antibodies). Like Omi-12, several members of this gene family have a glycosylation site at residue 102 of the heavy chain CDR3, the role of which is unclear. VH1-58 antibodies elicited during early pandemic or beta virus infection show reduced ability to neutralize Omicron e.g. mAb 253, Beta-47 and Known antibody D (AZD8895) show a reduction in activity of Omicron BA.1 vs Victoria respectively.
In contrast, Omi-12 has adapted and can potently neutralize Omicron and all VoCs (
Interestingly, in BA.2 the three serine residues mutated in BA.1 RBD: S371L, S373P and S375F in the loop adjacent to the lipid binding pocket are also muted in BA.2 but the mutation at 371 is to a Phe, which means that this is likely a single point mutation from early pandemic, whereas the S317L mutation in BA.1 requires two mutations. BA.2 may therefore have features common to earlier versions of the Omicron lineage. In addition, the various views provided of this part of the structure show that it adopts a range of different conformations. This is likely due to different crystal contacts and reflects flexibility in this loop region. This is likely to have a biological function since the Ser mutation required a double codon change and may possibly affect the presentation of the RBDs. Since we have multiple views of this loop in early pandemic virus, VoC, Omicron BA.1 and BA.2 we can see that flexibility is maintained across all variants. Modelling of effects on selected commercial known antibodies, early pandemic and Beta mAb for BA.1, BA.1.1 and BA.2 changes
Known Antibody A (REGN 10987) and B (10933): Known Antibody B (REGN 10933) binds the back of the left shoulder and REGN 10987 the right shoulder. Activity of both is knocked out by the Omicron lineage apart from Known Antibody A (REGN 10987) with BA.2. Known Antibody B (REGN 10933) H2 contacts residue 493 and since Q493R is present in all Omicron strains, neutralizing activity to Omicron is universally lost. Known Antibody A (REGN 10987) H2 contacts residue 446. BA.2 uniquely lacks the G446S mutation thus regn10987 retains some neutralization capability.
Known Antibody C (AZD1061) & D (AZD8895): Known antibody C (AZD1061) and D (AZD8895) bind the back of the left shoulder and the front of the right shoulder respectively both show reduced neutralization. Known antibody C (AZD1061) is still able to neutralize BA.2 and BA.3 (˜10-fold reduction) but neutralization of BA.1 is reduced >100-fold compared to Victoria and BA.1.1 >1000-fold compared to Victoria. Known antibody C (AZD1061) is affected due to contacts with G446S (absent in BA.2 and BA.3) and R346K (BA.1.1) mutations (contacted by L2 and H3). Known antibody D (AZD8895) is a VH1-58 antibody and contacts residues 477 (H3) & 493 (H2) and is compromised by the S477N and Q493R mutations universally present in the Omicron lineage. Known Antibody E (AZD7442) (a combination of C and D) maintains some neutralizing activity against Omicron strains as the sum of its components.
Known Antibodies F, G and H: All of Known Antibodies F, G and H suffer considerable loss of activity against Omicron. Activity of Known Antibodies F and H are completely lost whilst the activity of Known Antibody G (ADG20) on Omicron is reduced 276-fold
Known Antibodies I and J: Activity of both antibodies on the entire Omicron lineage is knocked out. Known Antibody J (Ly-CoV16) (VH3-53) makes extensive interactions with N501 and Y505 via L1 and L3 making it sensitive to mutations at these residues. Known Antibody I (Ly-CoV-555) is vulnerable to the E484K mutation in delta but likely tolerates E484A however, it also contacts residue 493, thus the universal
Omicron Q493R mutation will abrogate binding across the board.
Known Antibody K (S309): Known Antibody K (S309) retains reasonable activity across the Omicron lineage. S309 binds on the right flank with H3 contacting G339 and N343 glycans the latter close to the Serine 371, 373 and 375 mutations. The S371F mutation in BA.2 as opposed to S371L) may affect binding resulting in the slightly weaker activity with this virus.
The affinity of Omicron BA.1, BA.1.1, BA.2 and BA.3 RBDs for ACE2 was measured by SPR and BLI. The affinity of BA.1 was on a par with that of the early virus, 8 nM and 7 nM respectively (binding affinities for Omicron RBDs shown in Tables 14 and 18), implying that the increased affinity imparted by S477N, Q498R and N501Y is counter balanced by other mutations in the ACE2 footprint. The affinity of BA.2 was slightly increased compared to early virus (˜1.5-fold x and Y nM respectively). On the basis of earlier measurements of the contributions of individual mutations to binding affinity G496S and the triple-mutation S371L, S373P and S375F reduce binding by 2-fold and 2.2-fold respectively whereas BA.2 lacks G496S and has S371F. This may account for some of the difference but more likely the mutations in BA.2 on the edge of the ACE2 footprint may enhance binding. This is confirmed by the structure of the BA.2/ACE2.
The affinity of BA.4/5 RBD for ACE2 was also measured by SPR (
Surface plasmon resonance (SPR) was also used to characterise the interaction between ACE2 and the BA.2.75 RBD. The off-rate was very slow, leading to a sub-nanomolar affinity (BA.2.75/ACE2 KD=0.45 nM) (
BA.2+N460K RBD could not be expressed, but the binding affinity of BA.2+R493Q RBD to ACE2 (
The constellation of mutations in BA.2.75 compared to BA.2 have opposing effects on neutralization. The reversion mutation R493Q makes the virus easier to neutralize using vaccine serum (the vaccine contains Q493), whilst N460K reduces neutralization titres to a greater extent when expressed in isolation compared to the combination of mutations seen in BA.2.75. N460K is a novel substitution that has not appeared in preceding variants of SARS-CoV-2. This mutation was introduced into the BA.2 backbone and its impact on neutralisation by BNT162b2 serum was evaluated. Strikingly, BA.2+N460K titres were reduced 3.1-fold compared to BA.2, greater than the reduction seen with BA.2.75, and on a par with the reduction seen for BA.4/5.
Using a panel of potent mAbs derived from vaccinated individuals who suffered BA.1 vaccine breakthrough infection, it was shown that the activity of a number of mAbs belonging to the IGHV3-53/66 family are reduced or knocked out against BA.2.75. IGHV3-53/66 are the most frequently isolated mAbs in SARS-CoV-2, and bind an epitope on the ‘neck’. IGHV53/66 thus forms a major public antibody response and it is no surprise that the virus has evolved to escape this response.
Although BA.2+N460K RBD could not be expressed, a previous study using yeast display showed N460K can enhance RBD binding for ACE2, an effect similar to that seen with the N501Y mutation first described in Alpha (Zahradnik et al., 2021). Thus, N460K can both enhance antibody escape and increase receptor binding affinity.
Interestingly, BA.2.75 has also acquired the R493Q reversion (Q493R was acquired in BA.1 and present in all other Omicron sublineages except BA.4/5). BA.2.75 RBD was able to bind ACE2 with 9-fold higher affinity than BA.2 and more tightly than BA.4/5 (Dejnirattisai et al., 2022; Tuekprakhon et al., 2022). This is partly contributed by the R493Q mutation. BA.2.75 RBD has the highest receptor binding affinity among all SARS-CoV-2 variants measured to date.
These data suggest there may be a fine balance between antibody escape and ACE2 receptor affinity. Mutations in BA.2.75 lead to a reduction in neutralization titres of vaccine serum compared to BA.2. Individual BA.2.75 mutations can cause greater reduction in neutralization titres compared to the full BA.2.75 S sequence, but these are balanced by the R393Q reversion mutation, which may have been selected to increase affinity to ACE2 and increase the transmissibility of BA.2.75.
To evaluate the possible change in transmissibility of the BA.2 subvariants SPR experiments were performed to analyse their RBD binding to ACE2 (
The neutralization data above has been used to place BA.3 and BA.4/5 on an antigenic map. The method used for analysis of 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 individual viruses were independently modelled allowing for serum specific scaling of the responses. The measured and modelled responses are shown in
Neutralization of BA.2.75 was tested using serum from individuals previously infected during the course of the pandemic. These included serum obtained early in the pandemic (before the emergence of Alpha) together with serum obtained following Alpha, Beta, Gamma, Delta, BA.1 and BA.2 infection (
These data were used to place BA.2.75 onto a three dimensional antigenic map using the method previously reported in Tuekprakhon et al., 2022 (
As discussed in the detailed description above, antibodies derived from the same heavy chain V-gene may swap light chains to result in an antibody comprising the heavy chain variable region of a first antibody and a light chain variable region of a second antibody, and such new antibodies may have improved neutralisation and/or other characteristics when compared to the ‘parent’ antibodies.
Tables 4 to 12 provide examples of such antibodies that may be creased by swapping the light chain between antibodies derived from the same heavy chain V-gene. Table 17 provides information as to the heavy chain and light chain V-genes from which the 28 Omicron-specific mAbs are derived, together with their specificity to the RBD or NTD of the spike protein of SARS-CoV-2.
SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al, 2020), Alpha and Beta were provided by Public Health England, Gamma cultured from a throat swab from Brazil, Delta was a gift from Wendy Barclay and Thushan de Silva, from the UK G2P genotype to phenotype consortium and Omicron was grown from a positive throat swab (IRAS Project ID: 269573, Ethics Ref: 19/NW/0730. Briefly, VeroE6/TMPRSS2 cells (NIBSC) were maintained in Dulbecco's Modified Eagle Medium (DMEM) high glucose supplemented with 1% fetal bovine serum, 2 mM Glutamax, 100 IU/ml penicillin-streptomycin and 2.5 ug/ml amphotericin B, at 37° C. in the presence of 5% CO2 before inoculation with 200 ul of swab fluid. Cells were further maintained at 37° C. with daily observations for cytopathic effect (CPE). Virus containing supernatant were clarified at 80% CPE by centrifugation at 3,000 r.p.m. at 4° C. before being stored at −80° C. in single-use aliquots. Viral titres were determined by a focus-forming assay on Vero CCL-81 cells (ATCC). Sequencing of the Omicron isolate shows the expected consensus S gene changes (A67V, A69-70, T95I, G142D/A143-145, A211/L2121, 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.
Cells were infected with the SARS-CoV-2 virus using an MOI of 0.0001.
Virus containing supernatant were harvested at 80% CPE and spun at 3000 rpm at 4° C. before storage at −80° C. Viral titres were determined by a focus-forming assay on Vero cells. Victoria passage 5, Alpha passage 2 and Beta passage 4 stocks Gamma passage 1, Delta passage 3 and Omicron passage 1 were sequenced to verify that they contained the expected spike protein sequence and no changes to the furin cleavage sites.
Vero (ATCC CCL-81) and VeroE6/TMPRSS2 cells were cultured at 37° C. in Dulbecco's Modified Eagle medium (DMEM) high glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX (Gibco, 35050061) and 100 U/ml of penicillin-streptomycin. Human mAbs were expressed in HEK293T cells cultured in UltraDOMA PF Protein-free Medium (Cat #12-727F, LONZA) at 37° C. with 5% CO2. HEK293T (ATCC CRL-11268) cells were cultured in DMEM high glucose (Sigma-Aldrich) supplemented with 10% FBS, 1% 100× Mem Neaa (Gibco) and 1% 100× L-Glutamine (Gibco) at 37° C. with 5% CO2. To express RBD, RBD variants and ACE2, HEK293T cells were cultured in DMEM high glucose (Sigma) supplemented with 2% FBS, 1% 100× Mem Neaa and 1% 100× L-Glutamine at 37° C. for transfection. Omicron RBD and human mAbs were also expressed in HEK293T (ATCC CRL-11268) cells cultured in FreeStyle 293 Expression Medium (ThermoFisher, 12338018) at 37° C. with 5% CO2. E. coli DH5a bacteria were used for transformation and large-scale preparation of plasmids. A single colony was picked and cultured in LB broth at 37° C. at 200 rpm in a shaker overnight.
Sera from Pfizer Vaccinees
Pfizer vaccine serum was obtained from volunteers who had received either one or two doses of the BNT162b2 vaccine. Vaccinees were Health Care Workers, based at Oxford University Hospitals NHS Foundation Trust, not known to have prior infection with SARS-CoV-2 and were enrolled in the OPTIC Study as part of the Oxford Translational Gastrointestinal Unit GI Biobank Study 16/YH/0247 [research ethics committee (REC) at Yorkshire & The Humber—Sheffield] which has been amended for this purpose on 8 Jun. 2020. The study was conducted according to 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 were studied after receiving two doses of, and were sampled approximately 28 days (range 25-38), 180 days (range 178-221) and 270 days (range 243-273) after receiving two doses of Pfizer/BioNtech BNT162b2 mRNA Vaccine, 30 micrograms, administered intramuscularly after dilution (0.3 mL each), 17-28 days apart, then approximately 28 days (range 25-56) after receiving a third “booster dose of BNT162B2 vaccine. The mean age of vaccinees was 37 years (range 22-66), 21 male and 35 female.
Plasma from Early Pandemic and Alpha Cases
Participants from the first wave of SARS-CoV2 in the U.K. and those sequence confirmed with B.1.1.7 lineage in December 2020 and February 2021 were recruited through three studies: Sepsis Immunomics [Oxford REC C, reference:19/SC/0296]), ISARIC/WHO Clinical Characterisation Protocol for Severe Emerging Infections [Oxford REC C, reference 13/SC/0149] and the Gastro-intestinal illness in Oxford: COVID sub study [Sheffield REC, reference: 16/YH/0247]. Diagnosis was confirmed through reporting of symptoms consistent with COVID-19 and a test positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories. A blood sample was taken following consent at least 14 days after symptom onset. Clinical information including severity of disease (mild, severe or critical infection according to recommendations from the World Health Organisation) and times between symptom onset and sampling and age of participant was captured for all individuals at the time of sampling. Following heat inactivation of plasma/serum samples they were aliquoted so that no more than 3 freeze thaw cycles were performed for data generation.
Sera from Beta, Gamma and Delta and BA.1 Infected Cases
Beta and Delta samples from UK infected cases were collected under the “Innate and adaptive immunity against SARS-CoV-2 in healthcare worker family and household members” protocol affiliated to the Gastro-intestinal illness in Oxford: COVID sub study 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 disease occurring whilst in isolation and in direct contact with Beta/Delta sequence-confirmed cases. Additional Beta infected serum (sequence confirmed) was obtained from South Africa. At the time of swab collection patients signed an informed consent to consent for the collection of 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 conducted in accordance with Good Clinical Practice guidelines. Gamma samples were provided by the International Reference Laboratory for Coronavirus at FIOCRUZ (WHO) as part of the national surveillance for coronavirus and had the approval of the FIOCRUZ ethical committee (CEP 4.128.241) to continuously receive and analyse samples of COVID-19 suspected cases for virological surveillance. Clinical samples were shared with Oxford University, UK under the MTA IOC FIOCRUZ 21-02.
Sera from BA.1 Infected Cases, Study Subjects
Following informed consent, individuals with omicron BA.1 were co-enrolled into the ISARIC/WHO Clinical Characterisation Protocol for Severe Emerging Infections [Oxford REC C, reference 13/SC/0149] and the “Innate and adaptive immunity against SARS-CoV-2 in healthcare worker family and household members” protocol affiliated to the Gastro-intestinal illness in Oxford: COVID sub study [Sheffield REC, reference: 16/YH/0247] further approved by the University of Oxford Central University Research Ethics Committee. Diagnosis was confirmed through reporting of symptoms consistent with COVID-19 or a positive contact of a known Omicron case, and a test positive for SARS-CoV-2 using reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories and lineage sequence confirmed through national reference laboratories. A blood sample was taken following consent at least 10 days after PCR test confirmation. Clinical information including severity of disease (mild, severe or critical infection according to recommendations from the World Health Organisation) and times between symptom onset and sampling and age of participant was captured for all individuals at the time of sampling.
Full details of the randomized controlled trial of ChAdOx1 nCoV-19(AZD1222), were previously published (PMID: 33220855/PMID: 32702298). These studies were registered at ISRCTN (U.S. Pat. Nos. 15,281,137 and 89,951,424) and ClinicalTrials.gov (NCT04324606 and NCT04400838). A copy of the protocols was included in previous publications (Folegatti et al., 2020, Lancet 396, 467-478.).
Data from vaccinated volunteers who received two vaccinations are included in the Examples. Vaccine doses were either 5×1010 viral particles (standard dose; SD/SD cohort n=21) or half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort n=4). The interval between first and second dose was in the range of 8-14 weeks. Blood samples were collected and serum separated on the day of vaccination and on pre-specified days after vaccination e.g. 14 and 28 days after boost.
The neutralization potential of Ab was measured using a Focus Reduction Neutralization Test (FRNT), where the reduction in the number of the infected foci is compared to a negative control well without antibody. Briefly, serially diluted Ab or plasma was mixed with SARS-CoV-2 strains and incubated for 1 hr at 37° C. The mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplates containing confluent Vero cell monolayers in duplicate and incubated for a further 2 hrs followed by the addition of 1.5% semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit virus diffusion. A focus forming assay was then performed by staining Vero cells with human anti-NP mAb (mAb206) followed by peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, the foci (infected cells) approximately 100 per well in the absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate. Virus-infected cell foci were counted on the classic AID EliSpot reader using AID ELISpot software. The percentage of focus reduction was calculated and IC50 was determinedusing the probit program from the SPSS package.
Pseudotyped lentivirus expressing SARS-CoV-2 S proteins from ancestral strain (Victoria, S247R), BA.1, BA.1.1, and BA.2 were constructed as described before (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 insert fragments and pcDNA3.1 vector were cloned by using Gibson assembly. Victoria (S247R) construct is as previously described in Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B. 1.617 by vaccine and convalescent serum.” Cell 184.16 (2021): 4220-4236. To construct BA.1.1, mutagenic primers of R346K (R346K_F 5′-GTGTTCAATGCCACCAAATTCGCCAGCGTGTAC-3′ and R346K_R5′-GTACACGCTGGCGAATTTGGTGGCATTGAACAC-3′) were PCR amplified by using BA.1 construct as a template, together with two primers of pcDNA3.1 vector (pcDNA3.1_BamHI_F 5′-GGATCCATGTTCCTGCTGACCACCAAGAG-3′ and pcDNA3.1_Tag_S_EcoRI_R5′-GAATTCTCACTTCTCGAACTGAGGGTGGC-3′), purified by using QIAquick Gel Extraction Kit (QIAGEN) and followed by Gibson assembly. All constructs were verified by Sanger sequencing after plasmid isolation using QIAGEN Miniprep kit (QIAGEN).
A similar strategy was applied for BA.3 and BA.4/5, briefly, BA.3 mutations were constructed using the combination fragments from BA.1 and BA.2. The resulting mutations are as follows. The resulting mutations are as follows, A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211/L212I, G339D, S371F, S373P, S375F, D405N, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. Although BA.4/5 S protein shared some amino acid mutations with BA.2 (Nutalai et al., 2022), to generate BA.4/5 mutations were added A69-70, L452R, F486V, and R498Q. The resulting S gene-carrying pcDNA3.1 was used for generating pseudoviral particles together with the lentiviral packaging vector and transfer vector encoding luciferase reporter. Integrity of constructs was sequence confirmed.
The same method was also used to construct BA.2.12.1, and BA.2.75, by adding more mutations into the BA.2 construct. To generate BA.2.75, K147E, W152R, F157L, 1210V, G275S, G446S and N460K were added into a BA.2 backbone. 339D was also changed in BA.2 S into 339H, and 493R was reversed in BA.2 to 493Q as in the ancestral strain. To test single mutation impact, D339H, G446S, N460K and R493Q were introduced individually into a BA.2 backbone. The resulting pcDNA3.1 plasmid carrying S gene was used for generating pseudoviral particles together with the lentiviral packaging vector and transfer vector encoding a
The details of pseudoviral neutralization test were described previously (Liu, Chang, et al. “Reduced neutralization of SARS-CoV-2 B. 1.617 by vaccine and convalescent serum.” Cell 184.16 (2021): 4220-4236) with some modifications. Briefly, the neutralizing activity of potent monoclonal antibodies (mAbs) generated from donors who had recovered from Omicron- and Beta-infection as well as those who were infected during the early pandemic in UK were 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. A four-fold serial dilution of each mAb was incubated with pseudoviral particles at 37° C., 5% CO2 for 1 hr. The stable HEK293T/17 cells expressing human ACE2 were then added to the mixture at 1.5×104 cells/well. At 48 hr. post 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. The reaction was incubated at room temperature for 5 mins and the firefly luciferase activity was measured using CLARIOstar® (BMG Labtech, Ortenberg, Germany). The percentage of mAb neutralization was calculated relative to the control. Probit analysis was used to estimate the value of dilution that inhibits half of the maximum pseudotyped lentivirus infection (PVNT50).
To determine the neutralizing activity of convalescent plasma/serum samples or vaccine sera, 3-fold serial dilutions of samples were incubated with the pseudoviral particles for 1 hr and the same strategy as mAb was applied.
Cloning was done by using a restriction-free approach (Peleg and Unger, 2014). Mutagenic megaprimers were PCR amplified (KAPA HiFi HotStart ReadyMix, Roche, Switzerland, cat. KK3605), purified by using NucleoSpin® Gel and PCR Clean-up kit (Nacherey-Nagel, Germany, REF 740609.50) and cloned into pJYDC1 (Adgene ID: 162458) (Zahradnik et al., 2021a). Parental pJYDC1 molecules were 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 correctness of mutagenesis was verified by sequencing.
Expression plasmids of wild-type and Omicron BA. 1 spike and RBD of BA.1 and BA.2 were constructed encoding for 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 are the same as previously described (Dejnirattisai, Wanwisa, et al. “The antigenic anatomy of SARS-CoV-2 receptor binding domain.” Cell 184.8 (2021): 2183-2200). A synthetic codon-optimized RBD fragment of BA.2 was used as a template and construct was amplified by PCR and cloned into pNEO vector as previously described (Dejnirattisai et al., 2021a; Supasa et al., 2021; Zhou et al., 2021). The construct was verified by Sanger sequencing.
To generate H-is-tagged constructs of BA.4/5 RBD, site-directed PCR mutagenesis was performed using the BA.2 RBD construct as the template: (Nutalai et al., 2022), with the introduction of L452R, F486V and R493Q mutations, The gene fragment was amplified with pNeoRBD333Omi|F (5′ GGTTGCGTAGCTGAAACCGGTCATCACCATCACCATCACACC AATCTGTGCCCTTTCCGAC-3′) and pNeoRBD333_R (5′-TGATGGTGTGCTTGGTACCT TATTACTTCT TGCCGCACACGGTAGC-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 construct containing a BAP-His tag, the gene fragment was amplified with RBD333_F (5′-GCGTAGCTGAAACCGCCACCAATCTGTGC CCTTTCGAC-3′) and RBD333_BAP_R (5′-GTCATTCAGCAAGCTCTICTTGCCGCACACGG TAGC-3′), and cloned into the pOPINTTGneo-BAP vector (Huo et al., 2020, “Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2”. Nature structural & molecular biology 27, 846-854.). Cloning was performed using the ClonExpress II One Step Cloning Kit (Vazyme). The Constructs were verified by Sanger sequencing after plasmid isolation using QIAGEN Miniprep kit (QIAGEN).
To generate the BA.2.75 RBD construct, site-directed PCR mutagenesis was performed using the BA.2 Spike construct as the template (Nutalai et al., 2022), with the introduction of D339H, G446S, N460K and R493Q mutations suing primers listed in
Plasmids encoding RBDs were transfected into Expi293F™ Cells (ThermoFisher) by PEI, cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30° C. with 8% CO2 for 4 days. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich).
Plasmids encoding RBDs were transfected into Expi293F™ Cells (ThermoFisher) by PEI, cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 37° C. for 1 day followed by 30° C. for 3 days with 8% CO2. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare) through His-tag binding, followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.
Protein expression and purification were conducted as described previously (Dejnirattisai et al., 2021a; Zhou et al., 2020). Briefly, plasmids encoding proteins were transiently expressed in HEK293T (ATCC CRL-11268) cells. The conditioned medium was concentrated using a QuixStand benchtop system. His-tagged Omicron RBD were purified with a 5 mL HisTrap nickel column (GE Healthcare) and further polished 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). ˜4 mg of ACE2 was mixed with homemade His-tagged 3C protease and DTT (final concentration 1 mM). After incubated at 4° C. for one day, the sample was flown through a 5 mL HisTrap nickel column (GE Healthcare). His-tagged proteins were removed by the nickel column and purified ACE2 was harvested and concentrated.
IgG mAbs and Fab Purification
To purify full length IgG mAbs, supernatants of mAb expression were collected and filtered by a vacuum filter system and loaded on protein A/G beads over night at 4° C. Beads were washed with PBS three times and 0.1 M glycine pH 2.7 was used to elute IgG. The eluate was neutralized with Tris-HCl pH 8 buffer to make the final pH=7. The IgG concentration was determined by spectro-photometry and buffered exchanged into PBS. To express and purify Fabs 158 and EY6A, heavy chain and light chain expression plasmids of Fab were co-transfected into HEK293T cells by PEI. After cells cultured for 5 days at 37° C. with 5% CO2, culture supernatant was harvested and filtered using a 0.22 mm polyethersulfone filter. Fab 158 was purified using Strep-Tactin XT resin (IBA lifesciences) and Fab EY6A was purified with Ni-NTA column (GE HealthCare) and a 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 the antibodies heavy and light chains of the indicated antibodies were transiently transfected into 293Y cells and antibody purified from supernatant on protein A. Fab fragments of 58 and beta-55 were digested from purified IgGs with papain using a Pierce Fab Preparation Kit (Thermo Fisher), following the manufacturer's protocol.
The surface plasmon resonance experiments were performed using a Biacore T200 (GE Healthcare). All assays were performed with a running buffer of HBS-EP (Cytiva) at 25° C.
To determine the binding kinetics between the SARS-CoV-2 RBDs and ACE2/monoclonal antibody (mAb), a Protein A sensor chip (Cytiva) was used. ACE2-Fc ormAb was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilutions, at a flow rate of 30 μl min−1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
To determine the binding kinetics between the SARS-CoV-2 Spikes and ACE2, a CM5 sensor chip was used. The sensor chip was firstly activated by an injection of equal volume mix of EDC and NHS (Cytiva) at 20 uL/min for 300 s, followed by an injection of Spike sample at 20 μg/mL in 10 mM sodium acetate pH 5.0 (Cytiva) onto the sample flow cell of the sensor chip at 10 uL/min, and finally with an injection of 1.0 M Ethanolamine-HCl, pH 8.5 (Cytiva) at 20 uL/min for 180 s. The reference flow cell was left blank. ACE2 was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilutions, at a flow rate of 30 μl min−1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction.
All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1. To determine the binding kinetics between the RBDs and mAb Omi-32/Omi-42, a Biotin CAPture Kit (Cytiva) was used. Biotinylated RBD was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. The mAb Fab was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μl min-1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
To determine the binding affinity of BA.4/5 RBD and mAb Omi-12, a Protein A sensor chip (Cytiva) was used. The Ig Omi-12 was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over the two flow cells at a range of seven concentrations prepared by serial twofold dilutions, at a flow rate of 30 d min-1. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Prism9 (GraphPad).
To compare the binding profiles between BA.2 and BA.4/5 RBD for mAb Omi-06/Omi-25/Omi-26, a Protein A sensor chip (Cytiva) was used. mAb in the IgG form was immobilised onto the sample flow cell of the sensor chip to a similar level (˜350 RU). The reference flow cell was left blank. A single injection of RBD was performed over the two flow cells at 200 nM, at a flow rate of 30 d min-1. Running buffer was also injected using the same programme for background subtraction. The sensorgrams were plotted using Prism9 (GraphPad).
To compare the binding profiles between BA.2 and BA.4/5 RBD for mAb Omi-02/Omi-23/Omi-31, a Biotin CAPture Kit (Cytiva) was used. Biotinylated BA.2 and BA.4/5 RBD was immobilised onto the sample flow cell of the sensor chip to a similar level (˜120 RU). The reference flow cell was left blank. A single injection of mAb Fab was performed over the two flow cells at 200 nM, at a flow rate of 30 μl min-1. Running buffer was also injected using the same programme for background subtraction. The sensorgrams were plotted using Prism9 (GraphPad).
To determine the binding kinetics between BA.2.75 or BA.2+R493Q RBD and ACE2, a Protein A sensor chip (Cytiva) was used. ACE2-Fc was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions, at a flow rate of 30 l min-1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
To confirm the binding kinetics between the BA.2.75 RBD and ACE2, a Biotin CAPture Kit (Cytiva) was used. Biotinylated ACE2 (bio-ACE2) was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. The BA.2.75 RBD was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μl min−1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
To determine the binding kinetics between the BA.2.75 or BA.2 RBD and mAbs, a Biotin CAPture Kit (Cytiva) was used. Biotinylated RBD was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. The Fab of Omi-18 or Omi-32 was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μl min−1 using a single-cycle kinetics programme. For the binding of Omi-20 for bio-BA.2 RBD, the Fab of Omi-20 was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μl min−1 using a single-cycle kinetics programme. For the binding of Omi-20 for bio-BA.2.75 RBD, the Fab of Omi-20 was injected over the two flow cells at a range of eight concentrations prepared by serial twofold dilutions, at a flow rate of 30 μl min−1. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
To compare the binding profiles between BA.2 and BA.2.75 RBD for mAb Omi-29, a Biotin CAPture Kit (Cytiva) was used. Biotinylated BA.2 and BA.2.75 RBD was immobilised onto the sample flow cell of the sensor chip to a similar level (˜110 RU). The reference flow cell was left blank. A single injection of mAb Fab was performed over the two flow cells at 1 μM, at a flow rate of 30 μl min−1. Running buffer was also injected using the same programme for background subtraction. The sensorgrams were plotted using Prism9 (GraphPad).
To compare the binding profiles between BA.2 and BA.2.75 RBD for mAb Omi-36, a sensor chip Protein A (Cytiva) was used. mAb Omi-36 in the IgG form was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. A single injection of RBD was performed over the two flow cells at 200 nM, at a flow rate of 30 μl min−1. Running buffer was also injected using the same programme for background subtraction. The sensorgrams were plotted using Prism9 (GraphPad).
IgG mAbs and Fabs Production
AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, Lilly and Adagio antibodies were provided by Adagio, LY-CoV1404 was provided by LifeArc. For the in-house antibodies, heavy and light chains of the indicated antibodies were transiently transfected into 293Y or 293T cells and antibody purified from supernatant on protein A as previously described (Nutalai et al., 2022). Fabs were digested from purified IgGs with papain using a Pierce Fab Preparation Kit (Thermo Fisher), following the manufacturer's protocol.
Statistical analyses are reported in the results and figure legends. Neutralization was measured by FRNT. The percentage of focus reduction was calculated and IC50 (FRNT50) was determined using the probit program from the SPSS package. The Wilcoxon matched-pairs signed rank test was used for the analysis and two-tailed P values were calculated on geometric mean values.
RBD proteins were deglycosylated with Endoglycosidase F1 before used for crystallization. Omicron BA.1-RBD was mixed with Omi-12 and beta-54 Fabs, separately, in a 1:1:1 molar ratio, with a final concentration of 7 mg ml-1. These complexes were separately incubated at room temperature for 30 min. Initial screening of crystals was set up in Crystalquick 96-well X plates (Greiner Bio-One) with a Cartesian Robot using the 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, Journal of Applied Crystallography 36, 308-314).
Crystals of BA.1-RBD/Omi-12/beta-54 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. Complex of BA.1-RBD/Omi-12/beta-54 was screen in Hampton Research Ammonium sulphate screen C2, containing 2.4 M (NH4)2SO4 and 0.1 M citric acid pH 5.0, but only crystals of Fab Omi-12 alone were formed in this condition.
Purified BA.2.75 RBD was deglycosylated with Endoglycosidase H1 and mixed with ACE2 in a 1:1 molar ratio, with a final concentration of 13.0 mg ml−1. Initial screening of crystals was set up in Crystalquick 96-well X plates (Greiner Bio-One) with a Cartesian Robot using the 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 BA.2.75 RBD-ACE2 complex were formed in Hampton Research PEGRx condition 2-25, containing 0.1% (w/v) n-Octyl-b-D-glucoside, 0.1 M Sodium citrate tribasic dihydrate pH 5.5 and 22% (w/v) PEG 3350. Diffraction data were collected at 100 K at beamline 103 of Diamond Light Source, UK, using the automated queue system that allows unattended automated data collection (https://www.diamond.ac.uk/Instruments/Mx/I03/I03-Manual/Unattended-Data-Collections.html).
Diffraction data were collected at 100 K at beamline 103 of Diamond Light Source, UK. All data were collected as part of an automated queue system allowing unattended automated data collection (https://www.diamond.ac.uk/Instruments/Mx/I03/I03-Manual/Unattended-Data-Collections.html). Crystals were pre-frozen by mounting in loops and soaked for a second in cryo-protectant containing 25% glycerol and 75% mother liquor. Diffraction images of 0.1° rotation were recorded on an Eiger2 XE 16M detector (exposure time from 0.018 s per image, beam size 80×20 m, 10% beam transmission and wavelength of 0.9762 Å). Data were indexed, integrated and scaled with the automated data processing program Xia2-dials (Winter, 2010, Journal of applied crystallography 43, 186-190; Winter et al., 2018, Acta Crystallogr D Struct Biol 74, 85-97). 360° of data was collected from a single crystal for each of the data sets.
Structures were determined by molecular replacement with PHASER (McCoy et al., 2007, J Appl Crystallogr 40, 658-674). VhVI and ChCl domains which have the most 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., 2020, Nature structural & molecular biology 27, 950-958) were used as search models for each of the current structure determination.
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 for all the structures. Due to the lower resolution, only rigid-body and group B-factor refinement were performed for structures ofBA.1-RBD/O-12/Beta-54 complex.
Data collection and structure refinement statistics are given in Tables 19 and 25. Structural comparisons used 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 prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
The structure of the BA.1 RBD/Fab Omi-12/Fab Beta-54 ternary complex was determined to 5.5 Å resolution (Table 19,
Omi-12 and Antibody 253 are both derived from the germline heavy chain IGHV1-58. Interestingly, similar to antibody 253, other antibodies derived from the germline heavy chain IGHV1-58 described herein, i.e. Beta-47, Beta-25, antibody 55, antibody 165 and antibody 318, also has a valine (V) at position 53 in the heavy chain variable region, i.e. valine (V) at position 53 in SEQ ID NO: 262 (antibody 253), SEQ ID NO: 591 (Beta-47), SEQ ID NO: 461 (Beta-25), SEQ ID NO: 62 (antibody 55), SEQ ID NO: 182 (antibody 165) and SEQ ID NO: 332 (antibody 318). Position 53 in these sequences corresponds to position 58 according to IMGT numbering. Based on the data, modification of any of these antibodies by substitution of valine at position 53 with proline (i.e. V53P absolute numbering, or V58P according to IMGT numbering) would result in an antibody that would be effective against Omicron.
Furthermore, antibody AZD8895 (heavy chain variable region amino acid sequences provided in SEQ ID NO: 963 and light chain variable region amino acid sequence provided in SEQ ID NO: 965) is also derived from the germline heavy chain IGHV1-58 (e.g. see Dong et al. Nat Microbiol 6, 1233-1244 (2021)). AZD8895 has an isoleucine (I) at position 53 in the heavy chain variable region, which corresponds to position 58 according to IMGT numbering. Based on the data herein, modification of the heavy chain variable region AZD8895 (SEQ ID NO: 963) by substitution of isoleucine at position 53 with proline (i.e. 153P) using absolute numbering, or I58P using IMGT numbering, would result in an antibody that would be effective against Omicron.
Hence, the data indicate that the modification of VH1-58 antibodies such that a proline is present at position 53 (corresponding to position 58 according to IMGT numbering) in the heavy chain variable region would make them particularly effective against Omicron.
To elucidate the molecular mechanism for high affinity, the structure of the BA.2.75 RBD with ACE2 was determined by crystallography (according to the methods described in Example 4). As expected the binding mode was essentially indistinguishable from that observed before (
bValues in parentheses are for highest-resolution shell.
cRigid body and group B-factor refinement only.
aValues in parentheses are for highest-resolution shell.
Amino acid sequence of heavy chain and light chain variable regions of selected antibodies
Nucleotide sequence of heavy chain and light chain variable regions of selected antibodies
Amino acid sequences of CDRs
Amino acid sequence of heavy chain and light chain variable regions of selected antibodies
Nucleotide sequence of heavy chain and light chain variable regions of selected antibodies
Amino acid sequences of CDRs
Amino acid sequence of heavy chain and light chain variable regions of selected antibodies
Nucleotide sequence of heavy chain and light chain variable regions of selected antibodies
Amino acid sequences of CDRs
| Number | Date | Country | Kind |
|---|---|---|---|
| GB2202232.1 | Feb 2022 | GB | national |
| GB2203423.5 | Mar 2022 | GB | national |
| GB2206777.1 | May 2022 | GB | national |
| GB2212470.5 | Aug 2022 | GB | national |
| GB2214036.2 | Sep 2022 | GB | national |
| GB2215418.1 | Oct 2022 | GB | national |
| GB2301959.9 | Feb 2023 | GB | national |
This application is a continuation of U.S. application Ser. No. 18/171,216 (filed Feb. 17, 2023) which claims priority to GB Application No. 2202232.1 (filed Feb. 18 2022), GB Application No. 2203423.5 (filed Mar. 11, 2022), GB Application No. 2206777.1 (filed May 9, 2022), GB Application No. 2212470.5 (filed Aug. 26, 2022), GB Application No. 2214036.2 (filed Sep. 26, 2022), GB Application No. 2215418.1 (filed Oct. 18, 2022), and GB Application No. 2301959.9 (filed Feb. 10, 2023), each of which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18171216 | Feb 2023 | US |
| Child | 18664079 | US |
