An antibody or antigen binding fragment

Abstract

The present invention relates to an antibody molecule or an antigen binding fragment, which targets a SARS-related coronavirus, for example SARS-CoV-2 and variants thereof, suitably which is bovine derived. The invention further relates to a method of producing these antibody molecules and / or antigen binding fragments, and the uses of these antibody molecules and / or antigen binding fragments. The invention also relates to nucleic acid molecules encoding the molecules and / or fragments, and expression vectors and hosts cells comprising the same.

Description

[0001] AN ANTIBODY OR ANTIGEN BINDING FRAGMENT

[0002] Field of the Invention

[0003] The present invention relates to an antibody molecule or an antigen binding fragment, which targets a SARS-related coronavirus, for example SARS-CoV-2 and variants thereof. The invention further relates to a method of producing these antibody molecules and / or antigen binding fragments, and the uses of these antibody molecules and / or antigen binding fragments. The invention also relates to nucleic acid molecules encoding the molecules and / or fragments, and expression vectors and hosts cells comprising the same.

[0004] Background to the Invention

[0005] Since its emergence in 2019, SARS-CoV-2, the causative agent of COVID-19, has spread worldwide and has claimed more than 6.9 million lives. Adaptive immunity (both neutralising antibodies (nAbs) and T cell-mediated immunity) plays a crucial role in protection against severe disease and clearing virus, and this is induced following natural infection and immunisation. The introduction of vaccines against SARS-CoV-2 was a success, preventing 14 million associated deaths in the first year of the implementation of vaccination programmes globally. The majority of licenced vaccines for use in humans were developed using the ancestral Lineage A (Wuhan) SARS-CoV-2 spike (S) protein. However, the constant evolution of the virus has given rise to new variants of concern (VOCs), such as the Omicron variants, which have the potential to escape from antibodies in individuals with prior exposure to the virus and / or vaccination. Despite vaccination, immunocompromised patients often show impaired responses and develop severe disease, highlighting the need for therapeutic monoclonal antibodies with potent cross-reactivity. In addition, human cell tropism reported for many species within the Sarbecovirus subgenus highlights their potential for future spill-over events. Therefore, the development of broad- acting therapeutic interventions remains a high priority. Monoclonal antibodies (mAbs) against SARS-CoV-2 have been crucial in the development of effective treatment options against COVID-19. Broadly neutralising antibodies have been isolated from individuals who had recovered from COVID-19 and from mice. The first therapeutic mAb clinical trials started a few months after the release of the virus genome. The use of therapeutic mAbs remains important for the treatment of patients with severe disease and vulnerable populations such as elderly and immunocompromised individuals. However,the emergence of new variants, such as Omicron and its sublineages, has reduced the efficacy of the currently licenced therapeutic mAbs. Hence, the continued development and discovery of effective therapeutic mAbs against current and potential future variants remains essential.

[0006] Summary of the invention

[0007] In a first aspect, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2, or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response.

[0008] In an alternative embodiment, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 1-319 or 541-1208 of the Lineage A spike protein of SARS-CoV-2, or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response.

[0009] There is also provided an antibody molecule or antigen binding fragment which comprises an ultra-long bovine VHCDR3. The antibody molecule or antigen binding fragment may be as defined in any aspect herein.

[0010] In a second aspect, there is provided an antibody molecule or antigen binding fragment which reduces or inhibits binding of a SARS related coronavirus spike protein or a portion thereof (such as the RBD) to an ACE2 receptor. The antibody molecule or antigen binding fragment may be as defined in any aspect herein.

[0011] Suitably, an antibody molecule or antigen binding fragment of any aspect herein may neutralise at least 65% of the infectivity of SARS-CoV-2 Lineage A, and SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron (B.1.1.529) variants.

[0012] In a third aspect, there is provided an antibody molecule or an antigen binding fragment wherein the antibody molecule or antigen binding fragment comprises a bovine CDR-H3, or a fragment thereof, which has a sequence selected from the list consisting of SEQ ID NO. 3, 5, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51 or 54 of Table 1. Suitably, the antibody molecule or antigen binding fragment is isolated or recombinant.

[0013] Suitably, the antibody molecule or antigen binding fragment comprises a bovine ultra-long CDR-HR or a portion thereof which has a sequence selected from the list consisting of SEQ ID NO. 3, 5, 10, 12, 23, 37 or 40 of Table 1.Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 1, 6, 13, 16, 21, 24, 27, 30, 33, 38, 41, 46, 49 and 52 of Table 1; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of selected from the list consisting of SEQ ID NO. 2, 4, 7, 9, 11 , 14, 17, 19, 22, 25, 28, 31 , 3436, 39, 42, 44, 47, 50 and 53 of Table 1 ; and / or a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence selected from the list consisting of SEQ ID NO. 3, 5, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51, and 54 of Table 1.

[0014] Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) comprising a combination of heavy chain complementarity determining region 1 (VHCDR1), heavy chain complementarity determining region 2 (VHCDR2) and heavy chain complementarity determining region 3 (VHCDR3) selected from the combinations shown in Table 1, where each combination is represented by an antibody name (e.g P2, P7, 99, 101, 103 etc).

[0015] Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) selected from the list consisting of SEQ ID NO. 96-115 of Table 3, or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response.

[0016] Any of the above may comprise a light chain variable region (VL) or fragment thereof. A light chain variable region or fragment thereof may be selected from:

[0017] • a generic variable light chain, suitably which is capable of pairing with a VHCDR or VH as disclosed herein;

[0018] • a light chain variable region comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 55, 58, 61, 66, 69, 72, 75, 78, 80, 82, 84, 87, 90, and 93 of Table 2, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence selected from the list consisting of SEQ ID NO. 56, 59, 62, 67, 70, 73, 76, 85, 88, 91 and 94 of Table 2 and / or a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of selected from the list consisting of SEQ ID NO. 57, 60, 63, 64, 65, 68, 71, 74, 77, 79, 81, 83, 86, 89, 92 and 95 of Table 2; or

[0019] • a combination of light chain complementarity determining region 1 (VLCDR1), light chain complementarity determining region 2 (VLCDR2) and light chain complementaritydetermining region 3 (VLCDR3) selected from the combinations of SEQ ID Nos. 55, 56 and 57 (114); 58, 59 and 60 (113); 61, 62 and 63 (106, 68 and 115); 61, 62 and 64 (118); 61, 62 and 65 (105); 66, 67 and 68 (103); 69, 70 and 71 (101); 72, 73 and 74 (99); 75, 76 and 77 (73); 78, 73 and 79 (72); 80, 62 and 81 (66); 82, 76 and 83 (63); 84, 85 and 86 (60); 87, 88 and 89 (58); 90, 91 and 92 (54); or 93, 94 and 95 (53). (antibody / fragment names in brackets)

[0020] • A light chain variable region selected from the list consisting of SEQ ID NO. 116-133 of Table 4, or a fragment thereof.

[0021] Also provided is a fusion protein comprising an antigen binding fragment as defined herein, and a further polypeptide. The further polypeptide may be, without limitation, a toxin, a hormone, a therapeutic, or a diagnostic.

[0022] In a further aspect, there is provided a nucleic acid molecule encoding an antibody molecule or antigen binding fragment or a fusion protein according to any aspect of the invention.

[0023] In a further aspect, there is provided an expression vector comprising a nucleic acid molecule according to any aspect of the invention.

[0024] In a further aspect, there is provided a host cell comprising a nucleic acid molecule and / or expression vector according to any aspect of the invention.

[0025] In a further aspect, there is provided a pharmaceutical composition comprising an antibody molecule or antigen binding fragment, a nucleic acid molecule, an expression vector, a fusion protein, and / or a host cell according to any aspect of the invention; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0026] In a further aspect, there is provided a method of producing an antibody molecule or antigen binding fragment, a fusion protein or a nucleic acid molecule according to any aspect of the invention, the method comprising culturing a host cell according to any aspect of the invention under conditions suitable for gene expression.

[0027] In a further aspect, there is provided a method of producing an antibody molecule or antigen binding fragment or a fusion protein according to any aspect of the invention, comprising i) a step of immunizing a host animal with an amino acid sequence comprising amino acids 1-319 or 541-1208 and / or 319-541 of the Lineage A spike protein of SARS-CoV-2 as an antigen, ora fragment thereof which is capable of eliciting an SARS related coronavirus immune response; and ii) a step of producing a polyclonal or monoclonal antibody.Another aspect of the invention provides an antibody molecule or antigen binding fragment thereof produced by administering to a host animal an amino acid sequence comprising the amino acid sequence 1-319 or 541-1208 and / or319-541 of the Lineage Aspike protein of SARS-CoV-2 as an antigen, or a fragment thereof which is capable of eliciting an SARS related coronavirus immune response; and ii) a step of producing a polyclonal or monoclonal antibody.

[0028] In a further aspect, there is provided an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in therapy.

[0029] In a further aspect, there is provided an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in treating a viral infection, in a patient in need thereof. Suitably, the viral infection is a SARS-related coronavirus infection.

[0030] Suitably, the viral infection is caused by SARS-CoV-2 or a variant thereof.

[0031] In a further aspect, there is provided an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in preventing a viral infection, in a patient in need thereof. Suitably, the viral infection is a SARS-related coronavirus infection.

[0032] Suitably, the viral infection is caused by SARS-CoV-2 or a variant thereof.

[0033] In a further aspect, there is provided a method of diagnosing a viral infection in a subject, wherein the method comprises contacting a sample obtained from the subject with an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any aspect of the invention; and detecting binding of the an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention to any SARS-related coronavirus, suitably SARS-CoV-2 or variant thereof, which is present in the sample.

[0034] In a further aspect, there is provided a kit for detecting, treating or preventing a viral infection, wherein the diagnostic kit comprises an antibody molecule or antigen binding fragment, anucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any aspect of the invention.

[0035] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0036] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0037] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0038] Various aspects of the invention are described in further detail below.

[0039] Brief description of the Figures

[0040] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0041] Figure 1: (A) Schematic representation of the immunisation schedule. The process lasted for 242 days and it consisted of one prime immunisation with Lineage A S1 spike, two boosts with the same antigen, one boost with Lineage A full length spike and one boost with Lineage A trimeric spike. Blood was collected 4 and ~8 days post immunisation. (B) Neutralisation assay using cow serum from the final timepoint (D242) against pseudotyped SARS-CoV-2 variants and the original SARS-CoV. The serum was able to neutralise all pseudotypes tested and ID50 values were calculated. (C) Table showing the EC50 values (pg / mL) of spike binding mAbs against different forms of Lineage A spike (trimeric, full length, S1, RBD) and other variant spikes (Alpha, Beta, Omicron (BA.5)). The mAbs are divided in ultra-longs and non-ultra-longs based on their CDRH3 length in amino-acids. (D) Competition ELISA of the five RBD-binding mAbs (P2, P7, 99, 103, 101) with human ACE2 against Lineage A RBD. Antibodies 99, 103and 101 showed inhibition of ACE2 with IC50 values of 0.03, 0.32 and 0.45 pg / mL, respectively. Antibodies P2 and P7 showed partial inhibition. Mean and SD shown on graphs

[0042] Figure 2: (A) Single point neutralisations of the mAb panel against SARS-CoV-2 pseudotypes (Lineage A, Alpha, Beta, Gamma, Delta and Omicron). The antibodies were divided into ultralongs and non- ultra-longs according to their CDRH3 length in amino-acid. Antibodies able to neutralise all variants at 75% minimum were considered broadly neutralising and were investigated further. These antibodies were P2, P7, 105, 118 and 99. (B) Table with IC50 values (pg / mL) for the candidate mAbs against different SARS-CoV-2 pseudotypes (Lineage A, Alpha, Beta, Gamma, Delta, Omicron, BA5, XBB) and SARS-CoV-1. Antibody P7 neutralised all pseudotypes, including SARS-CoV-1. The second most broad neutralising antibody was 99, which neutralised all SARS-CoV-2 pseudotypes but not SARS-CoV-1. NN stands for “not neutralising” and N / A means that no experiments were performed for this mAb / pseudotype combination. (C) Table showing IC50 values (pg / mL) for antibodies P7 and 99 against SARS-CoV-2 live virus (Lineage A, Alpha, Beta, Delta, Omicron). Both antibodies neutralised every variant

[0043] Figure 3: EC50 values (pg / mL) of the five RBD-binding mAbs against a panel of 23 SARS-CoV-2 variants and other sarbecovirus RBDs. Any EC50 >2.5 pg / mL was disregarded. The most broadly reactive antibody was P7, which bound every RBD in the panel, with EC50 values ranging from 0.094 to 0.311 pg / mL. The rest of the antibodies bound 8 of 23, 7 of 23, 4 of 23 and 3 of 23 for 99, P2, 103 and 101, respectively. Next to the table is a phylogenetic tree of the corresponding sarbecovirus RBDs.Figure 4: In vivo challenge in Syrian hamsters. (A) Golden Syrian hamsters (n - 6 biologically independent animals per group) were infected intranasally with SARS-CoV-2 strain Delta; 104pfu. Individual cohorts were treated 24 h post- infection (hpi) with 200 pL of antibody intranasally (IN) or with PBS. Animals were monitored for weight loss at indicated time-points. Data are the mean value (n = 6) ± SEM. Comparisons were made using a repeated-measures two-way ANOVA with Geisser- Greenhouse's correction and Dunnett's multiple comparisons test; at day 7: PBS vs. mAb 99 10 mg / kg; **p = 0.0034, PBS vs. P7 10 mg / kg; ***p = 0.0109, PBS vs. 10 mg / kg Sotrovimab;**p = 0.0034 (B) RNA extracted from lungs was analysed for SARS-CoV-2 viral load using qRT-PCR for the N gene levels by qRT-PCR. Assays were normalised relative to levels of 18S RNA. Data for individual animals are shown with the mean value represented by a horizontal line. Data are mean value (n = 6) ±SEM and were analysed using a one-way ANOVA with Dunn's multiple comparisons test; PBS vs. 10 mg / kg mAb 99; p = 0.0006, PBS vs.

[0044] 10 mg / kg P7; p = 0.0008, PBS vs 10 mg / kg Sotrovimab; p = 0.0006. (C) RNA extracted from the nasal tissue was analysed for SARS-CoV-2 viral load using qRT-PCR for the N gene levels by qRT-PCR. Assays were normalised relative to levels of 18S RNA. Data for individual animals are shown with the mean value represented by a horizontal line. Data are mean value (n = 6) ±SEM and were analysed using a one-way ANOVA with Dunn's multiple comparisons test; PBS vs.10 mg / kg mAb 99; p = 0.0264, PBS vs. 10 mg / kg P7; p = 0. 0188, PBS vs 10 mg / kg Sotrovimab;p = 0. 0170

[0045] Figure 5: Histological changes in the lungs of male Syrian hamsters, aged 9-11 weeks, at day 7 post intranasal challenge with SARS-CoV-2 Delta at a dose of 104PFU that had received PBS (cohort 1), mAb 99 (cohort 2), mAB P7 (cohort 3) or Sotrovimab (cohort 4) 24 hours pre infection. Evidence of viral antigen expression is restricted to the animals from cohort 1. (A) Animal 1.5. The lung shows extensive multifocal consolidated areas (left image: arrows). The right image shows a higher magnification of a consolidated area, with hyperplastic bronchiolar epithelial cells (arrows), large activated type II pneumocytes (arrowheads) and embedded aggregates of neutrophils (small arrows). Viral antigen expression is restricted to very rare individual macrophages (left image: left inset, arrow) and rare alveoli with a few positive epithelial cells (left image: right inset, arrow). (B) Animal 2.6. The lung does not exhibit any histological changes. (C) Animal 3.6. The lung exhibits a few random focal consolidated areas (left: arrows). The right image shows a focal consolidated area with small aggregates of hyperplastic bronchiolar epithelial cells (arrows) and infiltrating leukocytes. (D) Animal 4.2. The lung does not exhibit any histological changes. HE stains. Bars = 1 mm (left column), 50 pm (right column: A) and 100 pm (right column: B-D). B: lumen of a bronchiole; V: lumen of a vein.Figure 6 - Gating strategy for PBMCs subjected to flow cytometry. (A) Viability gating with Annexin-V-FITC on the x-axis and Propidium Iodide on the y-axis. Cells in gate R2 were considered live and used for downstream sorting. (B) Sorting gate for SARS-CoV-2 spike double-positive live cells. X-axis represents APC stain and y-axis represents PE stain. Cells positive for both stains (gate R7) were sorted into a 96-well plate as single cells.

[0046] Figure 7. Binding curves of the eight ultra-long mAbs of the panel against different SARS-CoV-2 spike variants and forms of spike (Lineage A: trimer, full-length, S1, RBD, Alpha full-length, Beta full-length, Omicron trimer). The highest concentration of each mAb was 2.5pg / mL and the lowest was 3ng / mL

[0047] Figure 8. Binding curves of the 13 non-ultra-long mAbs of the panel against different SARS-CoV-2 spike variants and forms of spike (Lineage A: trimer, full-length, S1, RBD, Alpha full-length, Beta full-length, Omicron trimer). The highest concentration of each mAb was 2.5pg / mL and the lowest was 3ng / mL.

[0048] Figure 9. Neutralisation curves of antibodies P2, P7, 118, 105 (UL) and 99 (non- UL) against pseudotypes of SARS-CoV-2 variants.

[0049] Figure 10 Neutralisation curves of mAbs P7 (UL) and 99 (non-UL) against different variants of authentic SARS-CoV-2.

[0050] Figure 11. Binding curves of the RBD-binding mAbs against the RBD panel. Graphs show only the variants with detectable binding to the mAbs.

[0051] Figure 12 (A) Golden Syrian hamsters (n = 6 biologically independent animals per group) were infected intranasally with SARS-CoV-2 strain BA.5; 104pfu. Individual cohorts were treated 24 h post-infection (hpi) with 200 pl of antibody intranasally (IN) or with PBS. RNA extracted from lungs was analysed for SARS-CoV-2 viral load using qRT-PCR for the N gene levels by qRT-PCR. Assays were normalised relative to levels of 18S RNA. Data for individual animals are shown with the mean value represented by a horizontal line. Data are mean value (n = 6) ±SEM and were analysed using a one-way ANOVA with Dunn's multiple comparisons test; PBS vs. 4 mg / kg mAb 99; *p = 0. 0346, PBS vs. 4 mg / kg P7; **p = 0. 0096, PBS vs 4 mg / kg Sotrovimab; ns p = 0. 0856. (B) Golden Syrian hamsters (n = 6 biologically independent animals per group) were infected intranasally with SARS-CoV-2 strain LIVERPOOL; 104pfu. Individual cohorts were treated 24 h post-infection (hpi) with 200 pL of antibody intranasally (IN) or with PBS. Animals were monitored for weight loss at indicated time-points. Data are the mean value (n = 6) ± SEM. Comparisons were made using a repeated-measures two-way ANOVA with Geisser-Greenhouse's correction and Dunnett's multiple comparisons test; at day 7 there were no significant differences. (C) RNA extracted from the lung tissue was analysed for SARS-CoV-2 viral load using qRT-PCR for the N gene levels by qRT-PCR. Assays were normalised relative to levels of 18S RNA. Data for individual animals are shown with the mean value represented by a horizontal line. Data are mean value

[0052] (n = 6) ±SEM and were analysed using a one-way ANOVA with Dunn's multiple comparisons test; PBS vs. 4 mg / kg mAb 99; p = 0. 0007, PBS vs 4 mg / kg Sotrovimab; p = 0. 0007.

[0053] Detailed Description of the Invention

[0054] Bovines are known to produce antibodies with unique features, including very long heavy chain complementary determining region 3s (CDRH3s) averaging 26 amino acids and a subset of 'ultra- long' (UL) antibodies with CDRH3 lengths of up to 75 amino acids. An UL-CDRH3 forms a distinct extended conformation consisting of a stalk region and a cysteine-rich knob domain that mediates antibody-antigen complex formation (reviewed in K. Haakenson, R. Huang, V. V. Smider, Front Immunol 9, 1262 (2018)). Given the extended conformation of the UL-CDRH3 domain, it has been proposed that these antibodies could target epitopes of viral glycoproteins that are otherwise inaccessible to human B cell repertoires. Previous studies have demonstrated that immunisation of Bos taurus with soluble HIV Env trimers can elicit UL-CDRH3 broadly neutralising antibodies when tested against large panels of HIV isolates (B. Heydarchi et a!., Cell Reports Medicine 3, 100635 (2022)). Furthermore, surface display libraries generated from naive 8. taurus B-cells have shown that anti-SARS-CoV-2 spike paratopes exist within this B cell receptor (BCR) repertoire (M. J. Burke etal., Journal of Biological Chemistry 298, (2022)).

[0055] Bovine ultralong CDR-H3 have been characterized by a very unusual tri-dimensional structure comprising a “stalk domain” and a “knob domain”. The stalk domain is composed of two antiparallel p strands (each strand generally corresponding to about 12 residues). The knob domain is a disulfide rich domain which comprises a loop motif and sits atop of the stalk, which serves as a bridge to link the knob domain with the main bovine antibody scaffold. The CDR-H3 is derived from DNA rearrangement of variable (V), diversity (D), and joining (J) gene segments. The ultralong CDR-H3 may be encoded by IGHV1-7 and IGHD8-2 genes. An overview of Bovine Ultra Long CDRs is found at Haakenson et al, supra.

[0056] The present invention is based upon the identification of novel antibodies, and in particular novel ultra-long CDRs which show specificity for SARS-CoV-2 spike protein and variantsthereof. Novel antibodies and / or fragments thereof as described herein show SARS-CoV-2 antigen binding and / or neutralising activity.

[0057] Various aspects of the invention are described in further detail below.

[0058] Definitions

[0059] Herein, an “antibody” refers to an immunoglobulin protein, which is a protein consisting of one or more polypeptides encoded by immunoglobulin genes or fragments of immunoglobulin genes, and therefore having an immunoglobulin binding domain. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the immunoglobulin variable region genes. An antibody typically comprises two identical pairs of polypeptide chains where each may comprise a light chain (Ic) and a heavy chain (he). Light chains may be classified as being either kappa or lambda, and heavy chains may be classified as being gamma, mu, alpha, delta, or epsilon.

[0060] An "antibody light chain" or an "antibody heavy chain" refers to a polypeptide comprising the VL or VH, respectively. The VL is encoded by the minigenes V (variable) and J functional), and the VH by minigenes V, D (diversity), and J. Each of VL or VH includes the CDRs as well as the framework regions. Herein, antibody light chains and / or antibody heavy chains may, from time to time, be collectively referred to as "antibody chains." These terms encompass antibody chains containing mutations that do not disrupt the basic structure of VL or VH.

[0061] A "variable heavy" (VH) chain or a "variable light" (VL) chain (also termed VH domain or VL domain) refers to the polypeptide chains that make up the variable domain of an antibody. Heavy chain germline segments are designated as VH, DH and JH, and compilation thereof results in a nucleic acid encoding a VH chain. Light chain germline segments are designated as VL or JL, and include kappa and lambda light chains (VK and JK; VX and JX.) and compilation thereof results in a nucleic acid encoding a VL chain. It is understood that a light chain is either a kappa or lambda light chain, but does not include a kappa / lambda combination by virtue of compilation of a VK and JX.

[0062] A “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably, and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an fc region as defined herein.An “antibody fragment” as referred to herein is a molecule which is not a full antibody, but which comprises a portion of an antibody which binds to an antigen, suitably the antigen to which the full antibody binds.

[0063] The term “analyte,” as used herein, generally refers to a substance to be detected in a sample, for example an antigen.

[0064] An "antigen" refers to a predetermined antigen to which an antibody can selectively bind. An antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound.

[0065] By "selectively binds" or "specifically binds" means that an antibody or antibody fragment reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an antigen or an epitope than with alternative substances, including unrelated proteins or other antibodies / antibody fragments. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the terms “selectively binds” or “specifically binds”.

[0066] By "blocking" in relation to antibody activity means that the antibody or antibody fragment inhibits or reduces biological activity of the antigen to which it binds.

[0067] By “neutralising” activity means that the antibody or antibody fragment has the ability to fully or partially neutralise the biological effect of a pathogen. The ability to neutralise may be determined using methods available in the art, for example as described by urbanowicz etal., science translational medicine 13, eabj0847 (2021).).

[0068] By “monospecific” is meant an antibody or antibody fragment which binds to only one antigen of interest.

[0069] By “bispecific polypeptide” is meant an antibody or antibody fragment which binds to two antigens of interest.By “multi-specific polypeptide” is meant an antibody or antibody fragment which comprise at least two antigen binding domains, wherein the at least two antigen binding domains independently bind two different antigens or two different epitopes on the same antigen.

[0070] A “circulating immunoglobulin molecule” includes IgGI, lgG2, 1 gG3, lgG4, slgA, IgM and IgD, or a fragment of any thereof.

[0071] By "isolated" means that the molecule (e.g. antibody, antibody fragment, nucleic acid molecule, vector) has been separated (e.g. by purification means) from a component of its natural environment. In the context of the present disclosure, an "isolated" antibody or antibody fragment may be obtained from bovine, and optionally engineered to produce any variant according to the invention, or may be produced recombinantly or synthetically, for example by chemical synthesis.

[0072] The term "chimeric" refers to an antibody or antibody fragment comprising at least two portions, one being derived from a particular source or species, such as bovine, while the other portion is derived from a different source or species, such as human.

[0073] Herein, the term “engineered antibody” refers to an antibody which has been structurally modified to provide one or more altered characteristics compared to the native antibody, for example affinity, stability, activity, glycosylation etc.

[0074] The term “synthetic” refers to an antibody or antibody fragment that has been produced de novo by synthesis, notably by chemical synthesis as described in the present disclosure.

[0075] The term "single domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single domain antibodies include VH or VL or VHh or V-nar.

[0076] An “Fv” refers to two variable domains, for example co-operative variable domains, such as a cognate pair or affinity matured variable domains, i.e. A VH and VL pair.

[0077] A “single chain variable fragment” or “scFv” as employed herein refers to a single chain variable fragment comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) which is stabilised by a peptide linker between the VH and VL: variable domains. The VH and VL variable domains may be in any suitable orientation, for example thec-terminus of VH may be linked to the n-terminus of VL or the c-terminus of VL may be linked to the n-terminus of VH.

[0078] A "dsFv" refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.

[0079] An "fd fragment" refers to a fragment of an antibody containing one heavy chain portion of a f(ab')2 fragment, i.e. a variable domain (VH) and one constant region domain (chi) of an antibody heavy chain.

[0080] A "fab1fragment" refers to a fragment containing one half (one heavy chain and one light chain) of the f(ab')2 fragment.

[0081] A "f(ab')2 fragment" refers to bivalent antibody fragment composed of two Fab (antigenbinding) regions linked by disulfide bonds within the hinge region.

[0082] A "conjugate" refers to any molecule or moiety appended to another molecule.

[0083] The term “knob domain peptide” may be used to refer to an isolated antibody fragment as described in the present disclosure.

[0084] The term “dissociation constant” or “KD” refers to a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, such as when a complex falls apart into its component molecules. Equilibrium dissociation constants (KDs) can be determined using surface plasmon resonance. The lower the dissociation constant, the more tightly bound the component molecules, or the higher the affinity between the component molecules.

[0085] A “monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies, that is, for example, the individual antibodies comprising the population are identical and / or bind the same epitope(s)

[0086] A “nucleic acid molecule” refers to two or more nucleosides that are covalently linked together to form a polynucleotide chain.

[0087] A “protein” (used interchangeably with the terms “polypeptide,” and “peptide”) is a polymer of at least two amino acids covalently linked by an amide bond. A protein is a gene product,encoded by a coding sequence. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts.

[0088] Herein, the term “recombinant” may be used interchangeably with the terms “engineered” or “non-naturally occurring”, and refers to a form of a molecule (such as a protein, or nucleic acid molecule or cell) which has been modified in a manner that would not otherwise exist in nature, or has been produced or derived from a form which has been modified. A recombinant protein may be identical to a wild type protein, but may have been produced using recombinant techniques.

[0089] An "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0090] Detailed Description

[0091] Antibody binding

[0092] In a first aspect, the invention provides an antibody molecule, or an antigen binding fragment, that binds to i) the S1 protein (e.g. amino acids 1-674 of the Lineage A S protein); ii) the S protein ectodomain (e.g. amino acids 1-1211 of the Lineage A S protein); iii) the full length S protein (e.g. amino acids 1-1208 of the Lineage A S protein); and / or iv) the RBD (e.g amino acids 319-541 of the Lineage AS protein); and / or the corresponding sequence in a SAR-CoV-2 variant; ora fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response.

[0093] Suitably, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2, or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response. Suitably, the antibody molecule or antigen binding fragment may bind to a fragment of the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2 which comprises or consists of at least or no more than 5, 10, 15, 20, 25, 30, 40, 50, 70 or 100 consecutive amino acids of said sequence .

[0094] In an alternative embodiment, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 1-319 or 541-1208 of the Lineage A spike protein of SARS-CoV-2, or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response.There is also provided an antibody molecule or antigen binding fragment which comprises an ultra-long bovine VHCDR3. The antibody molecule or antigen binding fragment may be as defined in any aspect herein.

[0095] An ultra-long CDR may be 10-80 amino acids in length, suitably 20-75 amino acids in length. There is also provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2 or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response, and which binds to an RBD epitope in at least 3 SARS related coronaviruses selected from the group consisting of RMYN02, Yun11, RS4081, RF1, KHOSTA2, BTKY72, BM4831, SCH014, WIV1, LYRA3, CO28, RSSTT200, PANG17, RATG13, BA5.58, BA2.75.58, BQ1.1, BA2.75.2, XBB, BA2, and FK. Suitably, an antibody molecule or antigen binding fragment binds an RBD epitope in at least 4, 5, 6, 7, 8,, 9, 10 of aforementioned SARS related coronaviruses.

[0096] Suitably, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2 ora fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response, and which binds to an RBD epitope in at least 4 SARS related coronaviruses selected from the group consisting of RMYN02, Yun11, RS4081, RF1, KHOSTA2, BTKY72, BM4831, SCH014, WIV1, LYRA3, CO28, RSSTT200, PANG17, RATG13, BA5.58, BA2.75.58, BQ1.1, BA2.75.2, XBB, BA2, and FK, suitably binds to RSSTT200, PANG17, FKand Lineage A.

[0097] Suitably, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2 ora fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response, and which binds to an RBD epitope in at least 8 SARS related coronaviruses selected from the group consisting of RMYN02, Yun11, RS4081, RF1, KHOSTA2, BTKY72, BM4831, SCH014, WIV1, LYRA3, CO28, RSSTT200, PANG17, RATG13, BA5.58, BA2.75.58, BQ1.1, BA2.75.2, XBB, BA2, and FK, suitably binds to BM4831, RATG13, BA5.58, BA2.75.58, RSSTT200, PANG17, FKand Lineage A.

[0098] Suitably, there is provided an antibody molecule or an antigen binding fragment that binds to the amino acid sequence 319-541 of the Lineage A spike protein of SARS-CoV-2 ora fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response, and which binds to an RBD epitope in at least 6 SARS related coronaviruses selected from the group consisting of RMYN02, Yun11, RS4081, RF1, KHOSTA2, BTKY72, BM4831, SCH014,WIV1, LYRA3, CO28, RSSTT200, PANG17, RATG13, BA5.58, BA2.75.58, BQ1.1, BA2.75.2, XBB, BA2, and FK, suitably binds to BA2.75.2, PANG17, XBB, BA2, BA2.12.1, FKand Lineage A.

[0099] Most suitably, there is provided an antibody molecule or antigen binding fragment which binds an RBD epitope in all of the aforementioned SARS related coronaviruses.

[0100] Antibody function

[0101] In an aspect, there is provided an antibody molecule or antigen binding fragment which reduces or inhibits binding of the ACE2 receptor to a SARS related coronavirus spike protein or a portion thereof such as the RBD. The antibody molecule or antigen binding fragment may be as defined in any aspect herein.

[0102] Suitably, in inhibition of binding to theACE2 receptor, the antibody molecule or antigen binding fragment has an IC5o of 0.02 to 0.6pg / ml, suitably 0.03 to 0.56pg / ml.

[0103] Suitably, an antibody molecule or antigen binding fragment of any aspect herein neutralises at least 65% of the infectivity of SARS-CoV-2 Lineage A, and SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron (B.1.1.529) variants. Suitably, the antibody molecule or antigen binding fragment is capable of neutralizing at least 70, 75, 80, 85, 90 or 95% of the infectivity of SARS-CoV-2 Lineage A, and SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron (B.1.1.529) variants. Suitably, in neutralisation, the antibody molecule or antigen binding fragment has an IC5o of 0.001 to 1.5 pg / ml, suitably 0.001 to 0.5 pg / ml, suitably 0.001 to 0.3 pg / ml, or 0.01 to 1.5 pg / ml.

[0104] Antibody sequences

[0105] A third aspect of the present invention provides an antibody molecule or an antigen binding fragment wherein the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a fragment thereof which has a sequence selected from the list consisting of SEQ ID NO.

[0106] 3, 5, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51 or 54 of Table 1. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a fragment thereof which has a sequence of SEQ ID. NO. 3. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 5. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 8. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 26. Suitably, the antibody molecule or antigen bindingfragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 29. Suitably, the antibody molecule or antigen binding fragment is isolated or recombinant.

[0107] Suitably, the antibody molecule or antigen binding fragment comprises a bovine ultra-long CDR-HR or a portion thereof which as a sequence selected from the list consisting of SEQ ID NO. 3, 5, 10, 12, 23, 37 or 40 of Table 1. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 3. Suitably, the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 5. Suitably, a portion thereof has one or more of the same or substantially similar characteristics as the full length ultra-long CDR-HR, for example binding specificity, affinity, neutralisation ability and / or potency. A portion may be a knob portion of the ultra-long CDR-H3. The “knob” domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. The knob portion of an ultra-long CDR may be recognised as the cysteine rich portion capable of forming a loop, and following the disulphide bridge of the stalk.

[0108] Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 1, 6, 13, 16, 21, 24, 27, 30, 33, 38, 41, 46, 49 and 52 of Table 1; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of selected from the list consisting of SEQ ID NO. 2, 4, 7, 9, 11 , 14, 17, 19, 22, 25, 28, 31 , 3436, 39, 42, 44, 47, 50 and 53 of Table 1 ; and / or a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence selected from the list consisting of SEQ ID NO. 3, 5, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51, and 54 of Table 1.

[0109] Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) comprising a combination of heavy chain complementarity determining region 1 (VHCDR1), heavy chain complementarity determining region 2 (VHCDR2) and heavy chain complementarity determining region 3 (VHCDR3) selected from the combinations of: SEQ ID Nos. 1, 2 and 3 (P2); 1, 4 and 5 (P7); 6, 7 and 8 (99); 1, 9 and 10 (118), 1, 11 and 12 (115); 13, 14 and 15 (114), 16, 17 and 18 (113); 13, 19 and 20 (106), 21, 22 and 23 (105), 24, 25 and 26 (103), 27, 28 and 29 (101), 30, 31 and 32 (73), 33, 34 and 35 (72), 1, 36 and 37 (68), 38, 39 and 40 (66), 41, 42 and 43 (63), 13, 44 and 45 (60), 46, 47 and 48 (58), 49, 50 and 51 (53), and / or 52, 53 and 54 (54). Most suitably, the antibody molecule or antigen binding fragmentcomprises a heavy chain variable region (VH) comprising a combination of heavy chain complementarity determining region 1 (VHCDR1), heavy chain complementarity determining region 2 (VHCDR2) and heavy chain complementarity determining region 3 (VHCDR3) selected from the combinations of SEQ ID Nos. 1, 2 and 3 (P2); 1, 4 and 5 (P7); 6, 7 and 8 (99), 24, 25 and 26 (103), or 27, 28 and 29 (101).

[0110] Suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) selected from the list consisting of SEQ ID NO. 96-115 of Table 3. Most suitably, the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) having the sequence of SEQ ID NO. 96, 97, 98, 105 or 106.

[0111] Any of the above may comprise a light chain variable region (VL). A light chain variable region may be selected from:

[0112] • a generic variable light chain, for example of an IgL;

[0113] • a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of selected from the list consisting of SEQ ID NO. 57, 60, 63, 64, 65, 68, 71, 74, 77, 79, 81, 83, 86, 89, 92 and 95 of Table 2

[0114] • a light chain variable region comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 55, 58, 61, 66, 69, 72, 75, 78, 80, 82, 84, 87, 90, and 93 of Table 2, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence selected from the list consisting of SEQ ID NO. 56, 59, 62, 67, 70, 73, 76, 85, 88, 91 and 94 of Table 2 and / or a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of selected from the list consisting of SEQ ID NO. 57, 60, 63, 64, 65, 68, 71, 74, 77, 79, 81, 83, 86, 89, 92 and 95 of Table 2; or

[0115] • a combination of light chain complementarity determining region 1 (VLCDR1), light chain complementarity determining region 2 (VLCDR2) and light chain complementarity determining region 3 (VLCDR3) selected from the combinations of SEQ ID Nos. 55, 56 and 57 (114); 58, 59 and 60 (113); 61, 62 and 63 (106, 68 and 115); 61, 62 and 64 (118); 61, 62 and 65 (105); 66, 67 and 68 (103); 69, 70 and 71 (101); 72, 73 and 74 (99); 75, 76 and 77 (73); 78, 73 and 79 (72); 80, 62 and 81 (66); 82, 76 and 83 (63); 84, 85 and 86 (60); 87, 88 and 89 (58); 90, 91 and 92 (54); or 93, 94 and 95 (53).

[0116] • A light chain variable region selected from the list consisting of SEQ ID NO. 116-133 of Table 4.

[0117] A generic variable light chain may be a light chain of IgL or one or more of the CDRs thereof.Suitably, a variable light chain or fragment thereof pairs with the respective VHCDR or VH chain. Suitably, the variable light chain or fragment thereof is of a suitable length to pair the full length of the respective VHCDR or VH chain. Suitably, a VLCDR may be an ultra long CDR. An ultra long VLCDR may be from the same or different antibody to the VHCDR disclosed herein to which it is to be paired. An example of a suitable light chain may be the light chain of HIV-1 specific NC-1 antibody, suitably the VL thereof.

[0118] Suitably, the light chain domains or sequence(s) for each antibody molecule or antigen binding fragment is that which naturally pairs with the heavy chain domain or sequence, as indicated by the antibody molecule or antigen binding fragment name (e.g. P2, P7, 99 etc). Suitably, therefore, an antibody molecule or an antigen binding molecule may comprise a pair of VHCDR3 and VLCDR3 selected from the list of pairs shown in Table 5 (where a pair share the same molecule name).

[0119] Suitably, therefore, an antibody molecule or an antigen binding molecule may comprise a pair of heavy chain CDR(s) and light chain CDR(s) (CDR1, 2 and 3) selected from the list of pairs of heavy chain and light chain CDRs of Table 5 (where a pair of heavy chain and light chain CDRs share the same molecule name).

[0120] Suitably, an antibody molecule or an antigen binding molecule may comprise a heavy chain and a light chain, selected from the pairs of heavy chains and light chains listed in Table 6 (where a pair of heavy chain and light chain CDRs share the same molecule name).

[0121] Most suitably there is provided an antibody molecule or an antigen binding molecule comprising a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 3, and a VLCDR3 of an IgL.

[0122] Most suitably there is provided an antibody molecule or an antigen binding molecule comprising a bovine CDR-H3 or a portion thereof which has a sequence of SEQ ID. NO. 5, and a VLCDR3 of an IgL.

[0123] Most suitably there is provided an antibody molecule or antigen binding fragment comprising a combination of VH CDRs of SEQ ID Nos 6, 7 and 8 and VL CDRs of SEQ ID Nos. 72, 73 and 74, as shown in Table 5 (antibody molecule 99).Most suitably there is provided an antibody molecule or antigen binding fragment comprising a combination of VH CDRs of SEQ ID Nos 27, 28 and 29 and VL CDRs of SEQ ID Nos. 69, 70 and 71, as shown in Table 5 (antibody molecule 101).

[0124] Most suitably there is provided an antibody molecule or antigen binding fragment comprising a combination of VH CDRs of SEQ ID Nos 24, 25 and 26 and VL CDRs of SEQ ID Nos. 66, 67 and 68, as shown in Table 5 (antibody molecule 103).

[0125] The present invention includes any of the above mentioned antibody molecules or antigen binding fragments with at least 95%, 96%, 97%, 98% or 99% similarity or identity to the defined sequence.

[0126] In certain embodiments, a presently disclosed antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising amino acid sequences that are homologous or identical to the amino acid sequences of the antibodies or antigen binding fragments described herein, and wherein the antibodies or antigen-binding fragments thereof retain the desired functional properties of the antibodies or antigen-binding fragments thereof as described herein, for example antigen specificity, affinity, neutralization ability and potency, as defined herein.

[0127] A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine is considered to be a functionally equivalent residue to lysine.

[0128] In certain embodiments, the VHCDR1, VHCDR2 and / or VHCDR3 amino acid sequences, or the VLCDR1, VLCDR2 and / or VLCDR3 amino acid sequences, or the VH or VL amino acid sequences can be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous oridentical to the sequences set forth above. Such homologous or variant sequences can be obtained by mutagenesis (e.g., site- directed or PCR-mediated mutagenesis), followed by testing of the encoded altered antibody for retained function (i.e., the binding affinity) using the binding assays described herein.

[0129] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity or homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the nonlimiting examples below.

[0130] The percent homology or identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci (1988);14: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a P AMI 20 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol (1970);48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 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.

[0131] Additionally, or alternatively, the protein sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al., J Mol Biol (1990):215:403- 10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul etah, Nucleic Acids Res (1997);25( 17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0132] Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved, reduced, or abolished antibody-dependent cellular cytotoxicity (ADCC) or complement dependant cytotoxicity (CDC). In certain embodiments, a sequence disclosed herein, e.g., a CDR sequence, a VH sequence or a VL sequence, can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about six, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and / or substituted.

[0133] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, I g D, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively.

[0134] Virus and antigens

[0135] The SARS-CoV-2 spike (S) protein is a trimeric glycoprotein, which is located on the external surface of the virus. It consists of an extracellular N-terminus, a transmembrane domain which is anchored in the viral membrane and an intracellular C terminus. The N terminus comprises a signal peptide (amino acids 1-13), an S1 subunit n(amino acids 14-685) and an S2 subunit (amino acids 686-1273). The S1 subunit comprises a Receptor Binding Domain (RBD) at residues 319-541, which transits between an open and closed configuration. The RBD binds to the cell receptor, ACE2. The amino acid sequence of the SARS-CoV-2 spike protein is found at UniProt Reference Number P0TDC2.

[0136] Variants of a SARS related coronavirus (Sarbecoviruses) include Alpha, Beta, Gamma, Delta, and Omicron variants B.1.1.529 and BA.5 of SARS-CoV-2, and any other variants which arise and to which show at least 70% sequence identity on the spike protein sequence, suitably the RBD sequence, to one of the aforementioned viruses. Suitably, the sequence identity may be at least 75, 80, 85, 90 or 95%.

[0137] The antigen may suitably be a S protein, or a portion thereof. Suitably, the antigen may be i) the S1 protein (e.g. amino acids 1-674 of the Lineage AS protein); ii) the S protein ectodomain (e.g. amino acids 1-1211 of the Lineage A S protein); iii) the full length S protein (e.g. amino acids 1-1208 of the Lineage A S protein); and / or iv) the RBD (e.g amino acids 319-541 of theLineage A S protein); or the corresponding sequence in a SAR-CoV-2 variant; or any fragment thereof which is capable of elicting a SARS related coronavirus specific immune response. As per convention in the art, the numbering used herein is that of the Lineage A spike protein. The same numbering system is used in variants of SARS-CoV-2, where an insertion is preceded by the term “insert” and a deletion is preceded by the term “del”.

[0138] The skilled person is aware that not all residues within an epitope are always essential. A specific binding molecule may retain binding to an amino acid sequence with at least 70% identity to an epitope. The specific binding molecule may bind to any of the epitopes disclosed herein or an amino acid sequence having at least 70% identity thereto.

[0139] The epitope (part of the antigen to which the antibody or antigen binding fragment binds) may be at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or at least 35 amino acids in length. The epitope may be five to 20 amino acids in length. The epitope may be five to 15 amino acids in length. The epitope may be five to 12 amino acids in length. The epitope may be six to 12 amino acids in length. The epitope may be seven to 12 amino acids in length.

[0140] The epitope may be a continuous epitope or a discontinuous epitope. A continuous epitope may be any consecutive residues bound by the specific binding molecule. Consecutive residues are adjacent to one another in the primary structure of a polypeptide.

[0141] A discontinuous epitope may be any non-consecutive residues bound by the specific binding molecule. Discontinuous epitopes are typically formed by non-consecutive residues adopting nearby positions in three-dimensional space due to the folding of a polypeptide.

[0142] Alanine screening experiments can be used to identify specific epitope amino acid residues involved in antibody-antigen binding by substituting individual amino acids for alanine.

[0143] By “capable of elicting a SARS related coronavirus specific immune response” means that the fragment or portion of an antigen may result in the production of antibodies or immunoglobulins, in a host when exposed to the fragment or portion of the antigen.

[0144] Antibody structure

[0145] The term “antibody molecule” or “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies,polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired activity, such as binding specificity, neutralisation ability and / or potency.

[0146] In a natural antibody molecule, there are two heavy chains and two light chains. Each heavy chain and each light chain has at its N-terminal end a variable domain. Each variable domain is composed of four framework regions (FRs) alternating with three complementarity determining regions (CDRs). The residues in the variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated.

[0147] In some embodiments, the antibody molecule or antigen binding fragment may be a monoclonal antibody.

[0148] In some embodiments, the antibody molecule or antigen binding fragment may be a polyclonal antibody.

[0149] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.The term “antigen-binding fragment” or “antigen-binding region” of an antibody, as used herein, refers to that region or fragment of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., SARS-CoV-2 or a variant thereof). In some embodiments, the antigen binding fragment or “fragment” may be selected from the group consisting of a monovalent fragment consisting ofthe VL , VH , CL and CH1 domains, Fv, a Fab, a Fab’, Fab'-SH, a F(ab)z, a F(ab’)2, a single chain variable fragment (scFv), an antibody domain, a nanobody (VHH), a Shark VNAR, a minibody, a diabody, a linear antibody, an isolated complementarity determining region (CDR), an scFv-FC; and a multispecific antibody formed from antibody fragments and variations thereof.

[0150] An antigen binding fragment may as disclosed herein may comprise or consists of at least or no more than 5, 10, 15, 20, 25, 30, 40, 50, 70 or 100 consecutive amino acids of said sequence.

[0151] Suitably, the antibody molecule or antigen binding fragment is isolated. Suitably, it is isolated from a mammal which has been immunized with a SARS related coronavirus antigen, suitably a SAR-CoV-2 antigen, or an antigen of a variant thereof. It may be isolated from any suitable animal or host cell, for example a bovine, for example Bos Taurus.

[0152] Suitably, the antibody molecule or antigen binding fragment is recombinant.

[0153] An antibody molecule or antigen binding fragment may consist of any of the antibody molecules or binding fragments as disclosed herein.

[0154] A monovalent antibody is an antibody with affinity for one epitope, antigen, or strain of microorganism i.e. is an antibody fragment with one antigen-binding site.

[0155] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0156] Fab’ fragments are similar to Fab fragments but include some of the Fc portion or hinge of the antibody. Fab’ fragments are formed by reducing F(ab’)2 fragments and therefore contain a small portion of Fc. As opposed to F(ab) fragments, Fab’ fragments contain a free sulfhydrylgroup that can be alkylated or used in a conjugation with another compound of interest (such as a protein, toxin or dye). As used herein, “F(ab')2 ” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab1) (bivalent) regions, wherein each (ab1) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2 ” fragment can be split into two individual Fab’ fragments. As used herein, the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.

[0157] Single-chain variable fragments (scFv) are single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long.

[0158] Single-domain antibodies, also known as nanobodies, VHH, or hcAb are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No.

[0159] 6,248,516 B1).

[0160] A ’’Shark VNAR” is the isolated variable antigen receptor single domain antibody derived from naturally-occurring single-chain antibodies found in sharks and has several advantages over conventional monoclonal antibodies, such as improved thermostability.

[0161] A “minibody” is a class of bispecific fragments which are scFv-derived bispecific molecules. It is a bivalent fusion molecule with two scFvs fused to CH3. The scFv targeting antigen A is fused to the N-terminus of one of the CH3 domains and the scFv targeting antigen B to the other CH3.

[0162] VHH-Fc and scFv-Fc are two VHH and scFv antibody fragments respectively linked to an Fc region. scFv-Fc are around 50 kDa lighter than a full-size IgG antibody.

[0163] As understood by persons skilled in the art, the Fc fragment of an IgG is all of the lower base of the antibody's Y-shape, which includes a sulfhydryl-bridged hinge region and CH2 and CH3domains. The Fc fragment may be homodimeric. Cargo molecules may be attached to the hinge region via chemical conjugation to cysteine residues.

[0164] Fab fragments, scFv fragments, VHH fragments or protein fragments or domains may be linked to immunoglobulin CH3 domains to form a homodimer. Protein fragments or domains may be linked to the N-termini of an Fc fragment to form homodimers. Protein fragments or domains may be linked to the C-termini of an Fc fragment to form homodimers or to both the N- and C-termini of an Fc fragment to form homodimers.

[0165] Antibody fragments / antigen binding fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage).

[0166] In some embodiments, the antibody may be a full length antibody.

[0167] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0168] The antibody constant region domains of an antibody of the present disclosure, if present, for example in a full length antibody or a multi-specific molecule, may be selected having regard to the proposed function of the multi-specific 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. In particular, human IgG constant region domains may be used, especially of the lgG1 and lgG4 isotypes. The lgG1 constant region may be silenced lgG1.

[0169] “Fc-silenced” antibodies formats with varying degrees of reduced receptor binding have been developed with the introduction of mutations in the Fc of key amino acids that mediate Fc interactions with complement and Fc receptors. A common design feature of all these formats is that undesirable Fc interactions with Fey receptors and the complement receptor C1q can be decoupled from binding to the Neonatal Fc Receptor (FcRn) which conveys the sought after benefit of long serum persistence. As with effector binding, in vivo serum persistence conferred by FcRn is also a tunable property that can be modulated by mutations in the IgG Fc. “Silenced lgG1” refers to an IgG 1 constant region where the process of binding of immunoglobulin Fc toFc gamma receptors and complement protein C1 has been substantially reduced or eliminated, thus abolishing immune effector functions.

[0170] Fc region mutations may be included which alter the half life of the antibody or antigen binding fragment, for example by influencing binding to the neonatal Fc receptor. Suitable mutations may be those which increase the half life. Suitable mutations may include for example and without limitation M252Y / S254T / T256E (YTE) and M428L / N434S (LS), as well as T250Q / M428L, H433K / N434F, YD (M252Y / T256D), DQ (T256D / T307Q), and DW (T256D / T307W).

[0171] In one embodiment, the isolated antibody fragment or polypeptide according to the invention, is fused to one or more effector molecules, optionally via a linker, for example a cleavable linker. In one embodiment, the effector molecule is an antibody. In one embodiment, the effector molecule is a full IgG. In another embodiment, the effector molecule is selected from the list consisting of a Fab, a VHH, a VH, a VL, a scFv, and a dsscFv. In one embodiment, the effector molecule comprises an albumin binding domain. In one embodiment, the effector molecule is albumin ora protein comprising an albumin binding domain.

[0172] In some embodiments, the antibody or antibody fragment may comprise a heavy chain constant region selected from lgG1, lgG2, lgG3 and lgG4, preferably lgG1 and lgG4, most preferably lgG1.

[0173] In some embodiments, the antibody molecule or antibody fragment may comprise a heavy chain constant region of lgG2a. lgG2a is a murine subclass only.

[0174] In some embodiments, the antibody molecule comprises a light chain constant region of kappa.

[0175] In some embodiments, the antibody molecule is a rat antibody, a human antibody or a chimeric antibody.

[0176] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.A “chimeric antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by two or more species that utilizes two or more different species’ antibody repertoires.

[0177] In some embodiments, the antibody molecule or antigen binding fragment is humanised, optimised, and / or de-immunized.

[0178] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Suitably, there may be provided an antibody molecule or antigen binding fragment comprising an ultralong CDR as disclosed herein, suitably that of SEQ ID NO. 5, 3, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51 or 54 of Table 1, and a human framework region. Suitably, the ultra long CDR may be the only VHCDR present in the humanized molecule / fragment.

[0179] Standard antibody humanization techniques include engineering of a non-human framework to appear more human-like or adding non-human antibody features to a human framework. The non-human framework technique involves the comparison of amino acid sequences of a parent non-human antibody to the amino acid sequences of a similar human antibody. Subsequently, the amino acid sequence of the parent non-human antibody is altered such that its framework regions are more similar in sequence to the equivalent framework regions of the similar human antibody. In order to gain good binding capacities, development efforts need to be made for each antigen-binding sequence individually. The human framework technique involves all six murine CDRs being transferred onto a human antibody framework. The human framework technique involves the comparison of framework amino acid sequences of a parent non-human antibody to the framework amino acid sequences from a library of framework sequences from human antibodies. The comparisons are made using the sequence alignment techniques described herein. A human framework sequence that is similar to that of the parent non-human antibody framework is selected. Similar sequences may be defined as the sequence with the highest % sequence identity. These CDR-grafted antibodies generally do not retain their original affinity for antigen binding and certain non-human framework residues must beincorporated into the variable domains to support structural conformation of the grafted CDRs and restore antigen binding and affinity. Many of the framework positions that are likely to affect affinity have been identified, thus structural modelling to select new residues in a stepwise fashion can generally lead to variants with restored antigen binding. Alternatively, phage antibody libraries targeted at these residues can also be used to enhance and speed up the affinity maturation process.

[0180] “Optimised” as employed herein is intended to refer to where one, two, three, four, five or more amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is / are retained.

[0181] In some embodiments, the antibody molecule or antigen binding fragment has undergone affinity maturation.

[0182] Antibody affinity maturation is a process by which antibodies gain increased affinity, avidity, and anti-pathogen activity. Standard antibody affinity maturation techniques include two mutagenesis strategies: stochastic and targeted mutagenesis. In stochastic mutagenesis, the sequences for the variable fragment can be mutated randomly through error-prone PCR or by introducing mutator bacterial strains. Targeted mutagenesis introduces mutations in predictable positions, in selected CDR and framework regions, and are also workable by window mutagenesis or site-directed mutagenesis.

[0183] “De-immunized” refers to a technology for location and removal of T-cell epitopes through the combined use of immunological and molecular biology techniques. In the case of deimmunization of antibodies, mutations to remove T-cell epitopes can generally be introduced without significantly reducing the binding affinity of the antibody. Typically, "deimmunized" antibodies are created with human constant regions and by expression of genes encoding these antibodies in mammalian cells.

[0184] In some embodiments, the antibody molecule or antigen binding fragment may be monospecific. In some embodiments, the antibody molecule or antigen binding fragment may be bispecific. In certain embodiments, bispecific antibodies or antigen binding molecules may bind to two different epitopes of SARS-CoV-2, suitably to two different epitopes of the spike protein. One of the epitopes may be within the RBD. A bispecific antigen biding fragment maybe an sc-Fv, a tanden scFv, a diabody, a nanobody or a dual affinity retargeting molecule (DART).

[0185] The term “binds to” refers to the process by which an antibody molecule or an antigen binding fragment interacts with and attaches to its target. An “antibody that competes for binding” or “antibody that cross-competes for binding” with a reference antibody for binding to an antigen, refers to an antibody that blocks binding of the reference antibody to the antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to the antigen in a competition assay by 50% or more. An exemplary competition assay is described in “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0186] Nucleic acid

[0187] In a further aspect of the invention there is provided a nucleic acid encoding an antibody molecule or antigen binding fragment of any aspect of the invention.

[0188] A “nucleic acid” as described herein (or “isolated nucleic acid”) refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0189] A nucleic acid molecule of an aspect herein may be a DNA, a RNA (e.g. mRNA), or a cDNA sequence.

[0190] A nucleic acid molecule encoding an antibody or antigen binding fragment, as described herein, may be operably linked to one or more regulatory elements. A regulatory element may be a transcriptional promoter, enhancer, pro-peptide, translational signal, replication origin, or transcriptional and translational termination signal.

[0191] A suitable promoter will be based upon the expression system in which the nucleic acid molecule is transcribed and translated. A promoter may be constitutive or may be inducible. An inducible promoter may be induced by, for example, tetracycline, heat, oxygen, steroids, metals such as zinc or cadmium, or cAMP, or presence or absence of a nutrient. A promoter may direct tissue or cell specific expression. A promoter may have a weak, moderate or stronglevel of expression. A promoter may be host specific or non-specific. A promoter may have tight regulation or may be a leaky promoter. A suitable promoter may be stable, such that it is not silenced during long term expression. The selection of a suitable promoter will depend on factors such as the expression system, desired yield, and functionality of the promoter.

[0192] By way of non-limiting example, for bacterial host cells, suitable promoters for directing transcription of a nucleic acid molecule of the present disclosure may include the promoters obtained from the E. coli lac operon, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated APL promoter, Streptomyces coelicolor agarase gene (dag A), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amy / W), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25) or phage derived T7 promoter. By way of non-limiting example, for yeast host cells, suitable promoters for expression in S. cerevisiae may include GAL1, GAL10, ADH1, TEF1 orTEF2HXT7, CUP1GPD1 , PHO5, MET25 and suitable promoters for expression in P Pastoris may include AOX1, GAP, FRLD1, YPT1, DAS1, or PDC1. By way of non-limiting example, for expression in a cell line, suitable promotors may include CMV, SV40, EF1a, UbC, PGK, Tet-ON / Tet-Off, Mifepristone-inducible promoter, metal inducible promoter, HSP, Cre-LoxP. By way of non-limiting example, suitable promoters for expression in a plant cell include by way of non-limiting example, CMV, ubiquitin promoters eg Ubi1 or Ubi10, actin promoters from rice, Rubisco small unit promoter, seed specific promoters for example napin, tubulin promoters, root specific promoters for example RolD or pAUX1, plant viral promoters for example TMV, CsVMV, and FMV.

[0193] A nucleic acid molecule of the third aspect may be codon optimized for expression in a particular system. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used forexpression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. It is understood that all codons need not be replaced to optimize the codon usage of the enzyme since the wild type sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the enzymes may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than90% of codon positions of the full length coding region. A nucleic acid molecule of the aspect may be codon optimised for expression in a bacterial cell, for example E. coli or Bacillus subtilis, or a fungal cell, for example Aspergillus niger, A. oryzae or Trichoderma reesei, a yeast cell for example P. pastoris or S. cerevisiae, a cell line for example CHO cells, BHK cells, HEK293 cells, NSO cells, SP2 / 0 cells, a plant cell for example N. benthamiana, an insect cell for example Sf9 or High Five.

[0194] Expression vector

[0195] Further provided herein is an expression vector comprising a nucleic acid according to any aspect of the invention.

[0196] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” The choice of expression vector is influenced by the choice of host expression system. Many expression vectors suitable for the expression of an enzyme as described herein are available and known to those of skill in the art.

[0197] The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage.

[0198] Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)). A vector may be a cloning vector. Suitable cloning vectors will be available in the art, and include without limitation plasmids, for example pcDNA3.1, pBR322, the pUC series including pUC18 or pUC19, pBluescript, pGEM-T or pGEM-T Easy, the pET series including pET-28a, pET-21, the pCR series for example pCR2.1 or pCR4-TOPO, the pZErO series for example pZErO-2, the pBAD series for example pBAD33, pRS series, pLKO.1; viral vectors derived from a virus selected from the group consisting of baculovirus, paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus, or bacteriophage, for example A (e.g. Agt10, Agt11 , AZAP, AEMBL3, AFIX), M13 (e.g M13mp18 orM13mp19), filamentous phage for examplef1 or fd phage derivatives, P1 or PAC, T7 phage, and Lambda DASH; BACs for example pBACe3.6, pBeloBACH, pECBACI, pTARBAC, pCCIBAC, plndigoBAC, RP11 BAG library, or pBAC-Lac; YACs for example pYAC3, pYAC4, pYAC-RC, pCIasper, pYACneo, H-YACormega-YAC; and mammalian or human artificial chromosomes (MAGs or HACs). A vector suitable for use in a cell free system may be a linear expression template.

[0199] A viral vector is preferably an AAV based viral vectors. A variety of AAV capsids have been described in the art. Methods of generating AAV vectors have also been described extensively in the literature (e.g., WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; U.S. Pat. No.

[0200] 7,588,772 B2). The source of AAV capsids may be selected from an AAV which targets a desired tissue. For example, suitable AAV may include, e.g., AAV9 (U.S. Pat. No. 7,906,111; US 2011-0236353-A1), rhIO (WO 2003 / 042397) and / or hu37 (US 7,906, 111B2; US20110236353). However, other AAV, including, e.g., AAV1, AAV2, AAV-TT, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV. PHP. B (or variants thereof) and others may also be selected.

[0201] An expression vector of the present disclosure may comprise one or more selectable markers, to facilitate selection and maintenance of the transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0202] The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and / or secretion of the product of the recombinant DNA in a selected host.

[0203] Host cell

[0204] For expression of an antibody or antigen binding fragment of any of the aspects, a nucleic acid molecule or vector of the present disclosure may be provided in an expression system, which may be a host cell or a cell-free system. Where an expression system is a host cell, it may be produced by transfection of a host cell with an expression vector comprising a nucleic acid molecule of the second aspect of the disclosure.

[0205] Therefore, in a fourth aspect of the present invention there is provided a host cell or cell free system comprising a a nucleic acid molecule or vector of the third aspect.The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0206] A host cell may be a prokaryotic cell (e.g. E coli), a plant cell, a yeast cell, or a eukaryotic cell, suitably a mammalian cell. A host cell for plasmid maintenance may be E. coli. A host cell for expression may be a mammalian cell line. A host cell may be a cell line, for example a mammalian cell line, such as HEK293, CHO, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC.

[0207] A host cell may be recombinant. The present disclosure may also provide a population of host cells, for recombinant manufacture of a fusion protein of the disclosure. Such a population may be homogenous (comprising one type of host cell) or may be heterogenous (comprising a mixture of two or more different types of host cells).

[0208] Compositions

[0209] In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, and / or a host cell according to any aspect of the invention; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0210] “Pharmaceutically acceptable carriers”, “pharmaceutically acceptable vehicles”, “diluents” or “excipients” are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such asEDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos.

[0211] 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0212] An “adjuvant” is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. It is an ingredient in a medicine that increases or modifies the activity of the other ingredients.

[0213] Method of antibody production

[0214] In a sixth aspect, the invention provides a method of producing an antibody molecule or antigen binding fragment according to any aspect of the invention, the method comprising culturing a host cell according to any aspect of the invention under conditions suitable for gene expression.

[0215] Recombinant proteins, such as antibodies, are conventionally generated by transfecting the recombinant DNA into a host cell, following which the host cells are cultured and the transfected DNA transcribed and translated. Different host cells can be chosen for recombinant protein production, the choice of which depends on the type of protein that needs to be generated, its functional activity and requisite yield. Culturing a host cell under conditions suitable for gene expression allows for the expression of the recombinant DNA in the host cell and production of the required recombinant proteins.

[0216] Therefore, there is provided a method for the production of an antibody or antigen binding fragment of any of the aspects described herein, wherein the method comprises:

[0217] a) transforming a host cell or a cell free system with a nucleic acid molecule as described herein;

[0218] b) maintaining the host cell or cell free system under conditions to allow expression of the nucleic acid molecule;c) optionally extracting the expressed antibody or antigen binding fragment from the host cell or cell free system; and

[0219] optionally, purifying the antibody or antigen binding fragment.

[0220] Any suitable method of extraction may be used, for example and without limitation, centrifugation, filtration, anion exchange chromatography, crystallization, and / or HPLC. Any suitable method for purification may be used, including without limitation, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography and affinity chromatography. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques, such as SDS page and Size Exclusion Chromatography (SEC). Alternatively, an antibody molecule or antigen binding fragment may be produced by a method comprising a step of immunizing a host animal with a SARS related coronavirus antigen, suitably a SAR-CoV-2 antigen, and a step of producing a polyclonal or monoclonal antibody.

[0221] Suitably, an antigen of a SARS-related coronavirus, suitably SARS-CoV-2 or a variant thereof is i) the S1 protein (e.g. amino acids 1-674 of the Lineage A S protein); ii) the S protein ectodomain (e.g. amino acids 1-1211 of the Lineage A S protein); iii) the full length S protein (e.g. amino acids 1-1208 of the Lineage AS protein); and / or iv) the RBD (e.g. amino acids 319-541 of the Lineage AS protein); or the corresponding sequence in a SAR-CoV-2 variant; or any fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response. Also included are antigens which have at least 90, 95, 96, 97, 98 or 99% sequence identify to any of the above sequences.

[0222] “Immunizing” means to introduce immunogenic antigens, capable of eliciting an immune response, into an organism that results in said organism becoming protected to the disease associated with the antigens introduced.

[0223] A “host animal” is the animal into which a biologic or biologies are introduced.

[0224] “Antigen” means any substance that causes the body to produce an immune response against that substance.PBMCs are peripheral blood mononuclear cells, including lymphocytes, monocytes, and dendritic cells.

[0225] Further antibodies of the invention can be generated by first identifying antibodies from naive or immunised antibody repertoires or antibody-fragment libraries using technologies such as single B-cell technologies or phage-displaying panning respectively, then screening the targetbinding antibodies identified in the previous step in microplate-based cell assays forthose that show binding affinity for a SARS related coronavirus antigen, suitably a SARS-CoV-2 or variant antigen (e.g the spike protein), and / or that have the ability neutralise SARS-CoV-2 or a variant thereof, and / or inhibit or block ACE2.

[0226] Conjugates

[0227] In another aspect, the invention provides an antibody-conjugate which comprises:

[0228] (a) an antibody molecule or antigen binding fragment according to any aspect of the invention; and (b) a coupling moiety coupled to the antibody molecule or antigen binding fragment.

[0229] The term "antibody-conjugate" as used herein refers to antibody-based conjugates that are configured to deliver a “conjugate”, “coupling moiety” or “cargo” to a cell.

[0230] The term as used herein refers to an antibody that includes a targeting component linked to a cargo component. Accordingly, the targeting component may include an antibody Fab fragment, an antibody variable domain, nanobody, or a protein that binds to a cell surface receptor or cell surface molecule. The cargo component may include a protein, one or more imaging labels, such as radiolabels, fluorescent molecules or other labelled molecules.

[0231] “Conjugated” or “coupled to” generally means joined or bound to another molecule. The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an scFv. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.As described herein, the terms “cargo component”, “cargo molecule”, “coupling moiety” and “conjugate” all refer to functional molecules bound to or conjugated to the antibody or antibody fragment of the invention, which have a functional use and may cause an effect in a target cell.

[0232] Cargo molecules as described herein can include any molecule having a function useful to cause an effect in a target cell. For example, in addition to cytotoxic drugs or imaging labels, additional cargo molecules could be radiolabels that kill cells through radiation damage i.e. can be used therapeutically rather than for imaging. Examples of such radiolabels are Yttrium (Y)-90 and Iodine (I)- 131. Additional types of cargo molecules are identifiable by skilled persons upon reading the present disclosure.

[0233] The antibody molecule or antigen binding fragment and the coupling moiety or conjugate may be linked by a covalent bond or may be non-covalently associated with each other.

[0234] A cargo component, cargo molecule, coupling moiety or conjugate may comprise a linker. “Linker” refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a coupling moiety.

[0235] Suitably, the coupling moiety may be a biologically active moiety, such as the core molecule or ion of a drug, and / or a diagnostic moiety i.e. any molecule that is used to aid in diagnosing, characterizing or identifying disease.

[0236] Suitably, the coupling moiety may comprise a drug.

[0237] The antibody conjugates of the presently disclosed subject matter can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity.

[0238] Therapeutic uses

[0239] The invention also provides an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in therapy. The therapeutic use may be for treating or preventing a disease. Suitably, the disease is an infectious disease (also referred to as an infection). Suitably, the disease is a viral infection. Suitably, the infection is a SARS related coronavirus infection, suitably a SARS-CoV-2 infection or an infection by a variant thereof.An antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of treating a subject.

[0240] Any of the antibody molecule or antigen binding fragment, nucleic acid, expression vector, host cell, and / or pharmaceutical composition according to any aspect of the invention described herein may be used in methods, e.g., therapeutic methods.

[0241] An antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of vaccinating a subject.

[0242] Any of the antibody molecule or antigen binding fragment, nucleic acid, expression vector, host cell, and / or pharmaceutical composition according to any aspect of the invention described herein may be administered using any suitable method to deliver them to a subject, such as a cell, a plurality of cells, or a multicellular organism, in particular an animal or a human, such as via injection, particularly intravenous, subcutaneous or intramuscular injection, among other methods identifiable by skilled persons.

[0243] The antibody molecule or antibody fragment of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0244] The antibody molecule or antigen binding fragment of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disease being treated or prevented, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody molecule or antigen binding fragment need not be, but is optionally formulated with one or more agentscurrently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody, or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0245] Suitably, in the uses and methods of the invention, the antibody molecule, the antigen binding fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered in a therapeutically effective amount. The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in a clinical improvement of disease.

[0246] For treatment, the appropriate dosage of an antibody molecule or antigen binding fragment of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease, the type of antibody molecule or antigen binding fragment, the severity and course of the disease, and whether the antibody molecule or antigen binding fragment is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody molecule or antigen binding fragment, and the discretion of the attending physician. The antibody molecule or antigen binding fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the infection, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg) of antibody or antibody fragment can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of symptoms occurs. One exemplary dosage of the antibody molecule or antigen binding fragment would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.The antibody molecule, antigen binding fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered at suitable time intervals, for example weekly or monthly.

[0247] The antibody molecule or antigen binding fragment of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody molecule or antigen binding fragment of the invention may be co-administered with at least one additional therapeutic agent.

[0248] An antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of treating an infection of SARS related coronavirus, for example SARS-CoV-2 or a variant thereof in a patient.

[0249] An infectious disease is an illness due to a pathogen or its toxic product, which arises through transmission from an infected person, an infected animal, or a contaminated inanimate object to a susceptible host.

[0250] As used herein the terms “treat”, “treating” or “treatment” refer to a clinical improvement of a disease. A clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the disease.

[0251] In some embodiments, effective treatment may be demonstrated by slowing or halting the progression of the disease in the subject, or reversing the disease. Suitably, the disease may be reversed partially, or completely. In some embodiments, complete reversal of the disease may be sufficient to result in curing of the disease, which may be eliminating the infection.

[0252] In the context of the present disclosure, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In suitable embodiments, subjects are mammals, particularly primates, especially humans. In suitable embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In certain embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. Herein, the terms, “patients” and “subjects” may be used interchangeably.In some embodiments, the antibody molecule or antigen binding fragment reduces or inhibits the ability of the virus to enter a host cell (for example by reducing or inhibiting ACE2 binding), or neutralizes the effect of the virus.

[0253] Suitably, the treatment may comprise stratification of patients. “Treatment stratification” refers to the process of systematically arranging or categorizing patients based on their health status and other factors.

[0254] In some embodiments, the patient has a viral infection, suitably a SARS related coronavirus infection, such as a SARS-CoV-2 or variant thereof infection, or is at risk of such an infection. Suitably, the patient is showing one or more symptoms of an infection of a SARS related coronavirus, including but not limited to fever, cough, shortness of breath, difficulty breathing, sore throat, congestion, runny nose, fatigue, muscle ache, body ache, headache, nausea, vomiting, loss of tase, and / or loss of smell. Suitably, the patient may have an acute infection or chronic symptoms (also known as long-Covid).

[0255] Diagnostic method

[0256] The present invention also provides a method for detecting a SARS related coronavirus (suitably SARS-CoV-2 or a variant thereof), for example in a method of diagnosing a subject. Suitably, the method comprises the use of antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention, in the detection of SARS related coronavirus (suitably SARS-CoV-2 or a variant thereof) in a sample obtained from a subject. The method may comprise contacting a sample obtained from a subject with an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention; and detecting binding of the antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a host cell, and / or a pharmaceutical composition according to any aspect of the invention to any SARS related coronavirus (suitably SARS-CoV-2 or a variant thereof) present in the sample. Suitably, the method may be any suitable antigen detection assay, such as an ELISA.

[0257] Suitably, the sample is a saliva sample, a blood sample, a respiratory excretion, or a urine sample.

[0258] Suitably, the method may be conducted using a kit of the present invention.Kits

[0259] The presently disclosed subject matter provides kits for treating or ameliorating a disease or disorder, and / or detecting SARS related coronavirus e.g. SARS-CoV-2 or a variant thereof. In certain embodiments, the kit comprises the antibodies and / or antigen binding fragments of the aspects disclosed herein.

[0260] A kit of the invention may additionally a sterile container which contains the antibodies and / or antigen binding fragments of the aspects disclosed herein; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0261] A kit may further comprise instructions for administering the antibodies or antigen-binding fragments thereof, or the composition disclosed herein to a subject. The instructions can generally include information about the use of the antibodies or antigen-binding fragments thereof, and the composition disclosed herein. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment and / or prevention of disease or disorder or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0262] A kit may additionally comprise one or more components for carrying out a method of detection or treatment as described herein, for example an ELISA. Such components may include a buffer (such as a reconstitution buffer, wash buffer, assay buffer, lysis buffer, and / or blocking buffer); a container (e.g., tube, microtiter plates, or strips) for conducting an assay; a calibrator or control; a reagent dispenser; a stabilizer; and the like. The relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. The reagents may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration.Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0263] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0264] Table 1 Heavy chain CDR sequences:

[0265]

[0266]

[0267]

[0268] Table 2: Light chain CDR sequences:

[0269] CDR Assignments (Light chain):

[0270]

[0271]

[0272] Table 3 Heavy chain sequences from which the CDRs of Table 1 are derived (VH PLUS PARTIAL J REGION):

[0273]

[0274]

[0275] Table 4 - light chain sequences from which the CDRs of Table 2 are derived (VL plus J)

[0276] >

[0277]

[0278] Table 5 Combinations of heavy and light chains

[0279]

[0280]

[0281]

[0282] Table 6 Heavy chain and light sequences

[0283]

[0284]

[0285]

[0286] SARS-CoV-2 spike protein sequence (SEQ ID NO. 134):

[0287] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS N VTWFHAI H VSGTNGTKRFDN PVLPFN DGVYFASTEKSNI I RGWI FGTTLDSKTQSLLI VN NATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD YSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYK LPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVE GFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNT SNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECD IPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEIL PVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVK QIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARD LICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGI GVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSN FGAISSVLN DI LSRLDKVEAEVQI DRLITGRLQSLQTYVTQQLI RAAEI RASAN LAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFP REGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKE ELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKW PWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHY T

[0288] Aspects of the invention are demonstrated by the following non-limiting examples.

[0289] EXAMPLES SARS-CoV-2 immunised cattle produce potent and broadly neutralising antibodies

[0290] To investigate bovine immune responses against SARS-CoV-2 spike, a series of immunisations were performed on two Holstein bullocks. The immunisation process lasted for 242 days and a total of five immunisations were performed: one prime immunisation and two boosts with the S1 subunit of Lineage A S1 spike (aa 1-674), one boost with Lineage A spike ectodomain (S1 and partial S2) and a final boost with Lineage A trimeric spike, (transmembrane domain replaced with a trimerization domain) (Fig. 1A). Sera and blood were collected 4- and 8-days after each boost. Sera binding was measured using an antigenbinding assay utilising spikes from different variants as target antigen. Maximum breadth and potency were observed in sera harvested at D242 post- immunisation (data not shown). Neutralisation assays using lentivirus-based pseudotypes supplemented with spikes representative of the original Lineage A SARS-CoV-2, variants of concern (Alpha, Beta, Gamma, Delta, Omicron variants B.1.1.529 and BA.5), and to test pan- species breadth, SARS-CoV-1 Frankfurt 1 strain yielded ID50 dilution values for Lineage A, Alpha, Beta, Omicron and Omicron BA5 of approximately 1:1000, and for Gamma, Delta and SARS-CoV-Fra1 1:100 (Fig. 1B).

[0291] Having established the presence of potent and broadly neutralising serum antibody responses at D242 from both animals, we then went on to generate a panel of spike-specific monoclonal antibodies. To achieve this, two methods were performed. The first utilised single-cell sorting of antigen-specific B cells in which peripheral blood mononuclear cells (PBMCs) from D242 were subjected to flow cytometry using a dead / live gate and Lineage A trimeric spike labelled with two different fluorochromes (fig. 6). Single B-cells specific for Lineage A trimer were sorted into 96- well plates and then PCR amplified using an IgG-specific nested primer PCR to retrieve individual heavy and light chain pairings. These were cloned into a human lgG1 expression cassette and expressed in mammalian cells. This yielded an initial panel of 31 candidate mAbs. The second method used a phage display approach in which antigen-specific B cells from D242 were isolated by flow cytometry using dual-staining with soluble 2P and GSAS substituted furin cleavage site trimeric spike protein. Isolated cells were pooled, RNA extracted, reverse transcribed and the resulting cDNA used as template in a nested primer PCR designed to preferentially amplify heavy chains derived from the VH1-7 germline, from which all ultra-long antibodies derive (M. L. Vadnais, V. V. Smider, Bos taurus ultralong CDR H3 antibodies. Curr Opin Struct Biol 38, 62-67 (2016).). Resulting PCR products were cloned, paired with a common light chain to generate Fab-p3-phage display libraries, which were then biopanned against SARS-CoV-2 Lineage A trimer followed by a second round of biopanning against Omicron BA.5 trimeric spike. This method identified a further two mAbs, P2 from round one and P7 from round 2, which were subcloned into a human I gG 1 expression cassette and expressed in mammalian cells and added to the panel. The length of the CDRH3 of each mAb in the panel ranged between 16-63 amino acids (aa). Ten of 33 antibodies were ultra-long with CDRH3 >50aa and they all encoded the same V-gene segment, VH1-7.

[0292] Isolated monoclonals show pan-sarbecovirus reactivity in vitro

[0293] In the next step, we characterised the reactivity and neutralising ability of the mAb panel. Twenty- one antibodies showed reactivity to Lineage A spike protein, eight of which wereultra-long (fig. 7) and 13 were non-ultra-long (fig 8). Nine mAbs bound to all variant spike proteins. Moreover, five antibodies were identified as RBD binders (99, 101, 103, P2 and P7) whereas the other mAbs bound outside the RBD (Fig. 1C). A competition ELISA assay was performed on the five RBD binding antibodies to assess whether they inhibit ACE2 receptor binding to Lineage A RBD spike. Antibody 99 showed the strongest inhibition of ACE2 (IC50 of 0.03 pg / mL), followed by antibodies 103 and 101 (IC500.32 pg / mL and 0.45 pg / mL, respectively). Antibodies P2 and P7 showed partial inhibition with a plateau of 70% ACE2 binding, suggesting accessibility hinderance to the RBD (Fig. 1D).

[0294] Having characterised the binding properties of each mAb in the panel, the next step was to assess their neutralisation potencies and breadth. To achieve this, the entire mAb panel was used in single- point neutralisation assays (1 pg / mL final mAb concentration) performed against pseudotyped SARS-CoV-2 viruses, including Lineage A, Alpha, Beta, Gamma, Delta and Omicron (B.1.1.529). Antibodies that were able to neutralise >75% infectivity of all variants were selected for further in-depth investigation. These criteria were fulfilled by four UL antibodies (P2, P7, 105, 118) and one non-UL (99) (Fig. 2A), the latter showing 100% neutralisation against all variants at 1 pg / mL.

[0295] These five candidate mAbs were subjected to further neutralisation experiments against pseudotyped SARS-CoV-2 viruses to determine IC50 values against different variants of concern (Fig. 2B). mAb 99 performed consistently well against all SARS-CoV-2 variants, including more contemporary variants such as Omicron BA.5 and XBB, with IC50 values ranging from 0.002 pg / mL (Gamma) to 0.22 pg / mL (XBB) (fig. 9). mAb P2 had comparable potency to mAb 99 with IC50 values ranging from 0.006 pg / mL (Gamma) to 0.2 pg / mL (Lineage A); however, did not neutralise variant Omicron BA.5 and SARS-CoV-1 (fig. 9). The non-RBD UL antibodies 105 and 118 had a similar breadth across the variants yet differed in potencies (fig. 9). The IC50 values for mAb 105 were between 0.003 pg / mL (Gamma) and 1.5 pg / mL (Omicorn BA.5) and for mAb 118 between 0.001 pg / mL (Omicron) and 1.5 pg / mL (Omicron BA.5). The antibody with the greatest neutralising breadth was P7, which was able to neutralise all variants tested, including the 2002 epidemic SARS-CoV strain, with IC50 values ranging from 0.01 pg / mL (SARS-CoV-1) to 1.06 pg / mL (XBB) (fig.

[0296] 9).

[0297] The two neutralising antibodies showing maximal potency and breadth, P7 and 99, were also tested against Lineage A, Alpha, Beta, Delta and Omicron variants in an authentic virus neutralisation assay. Both mAbs showed neutralising activity against all variants. The IC50 values for P7 ranged between 0.2 pg / mL (Delta) and 2.48 pg / mL (Lineage A), whereas for mAb 99, values ranged between 0.01 pg / mL (Alpha and Delta) and 0.56 pg / mL (LineageA) (Fig. 2C, fig. 10). Interestingly, both antibodies performed better against variants of concern (especially Delta) than against the original Lineage A, which the cattle were immunised with. Comparison between the pseudotype and the authentic virus assay showed similar IC50 values for 99, except for Lineage A which was less potent in the authentic virus assay. However, there was disparity between the two assays for P7, which consistently showed reduced neutralisation activity against all authentic virus variants tested. To determine the full breadth of the RBD-binding mAbs (P2, P7, 99, 103 and 101), binding assays were performed on a panel of 23 SARS-CoV-2 and other sarbecovirus RBD spike regions.

[0298] Remarkably, P7 showed strong RBD binding across the panel, with EC50 values ranging from

[0299] 0.083 pg / mL to 0.311 pg / mL, suggesting targeting of a conserved epitope within the RBD (Fig. 3). The second most broadly reactive antibody was mAb 99, followed by P2, binding 8 of 23 and 7 of 23 RBDs, respectively. Antibodies 103 and 101 were the least broad, binding 4 of 23 and 3 of 23 RBDs, respectively, and they showed similar binding patterns; all of the RBDs they bound were also bound by mAb 99 (fig. 11).

[0300] mAbs 99 and P7 protect in in vivo challenge model

[0301] To assess the efficacy of candidate mAbs in vivo, we performed authentic virus challenge experiments in Syrian hamsters. The study consisted of four groups of six animals that were challenged with the SARS-CoV-2 Delta (1 x 104pfu) on day 0, and then treated with mAb or placebo on day 1 , with weight changes followed over 7 days. The animals in the untreated group showed a significant and progressive weight loss (10% by day 7), whereas all animals treated therapeutically showed only a small weight loss 24 h after viral challenge, and from day 2 had recovered to at least pre-challenged weights (Fig. 4A). The weight loss in all mAb-treated groups was significantly different from the control group given PBS alone (p < 0.05 and p < 0.01 ; repeated measures two-way ANOVA with Dunnett's multiple comparison test). Analysis of viral load in the lungs at day 7 by qPCR for Nucleoprotein (N1) RNA showed a decrease in the mean value in treated compared to the untreated control animals (Fig. 4B). This decrease was significantly different between mAb 99 (2x103copies of N1 / pg of RNA; p = 0.0006), P7 (3x103copies of N1 / pg of RNA; p = 0.0008) and PBS only (4x104copies of N1 / pg of RNA). Similar results were observed for the commercial control Sotrovimab (7x102copies of N1 / pg of RNA; p = 0.0006; one-way ANOVA with Dunnett's multiple comparison). The measured viral load in the nasal tissue was also significantly reduced in the mAb treated animals. In mAb 99 treated (1x105copies of N1 / pg of RNA; p = 0.0264), P7 treated (4x104copies of N1 / pg of RNA; p = 0.0188), Sotrovimab treated (9x103copies of N1 / pg of RNA; p= 0.0170) and PBS only (2x106copies of N1 / pg of RNA; Fig. 4C). A histological and immunohistological examination was performed on the lungs of all animals in this experiment. The PBS control group, revealed changes consistent with SARS-CoV- 2 Delta infection in hamsters at this stage (L. Heydemann et al., Hamster model for post-COVID-19 alveolar regeneration offers an opportunity to understand post-acute sequelae of SARS-CoV-2. Nat Commun 14, 3267 (2023)), represented by multifocal consolidated areas with evidence of epithelial regeneration (represented by hyperplastic bronchiolar epithelial cells), type II pneumocyte activation and hyperplasia, and a predominantly mononuclear inflammatory infiltrate that still contained a proportion of neutrophils (Fig. 5A). There was very limited viral antigen expression, in rare individual macrophages in consolidated areas, and in a few alveolar epithelial cells (Fig. 5A). In all treated hamster cohorts, there was no evidence of viral antigen expression in any of the lung sections. In the mAb 99 treated cohort, four animals exhibited either no or minimal non-specific changes (perivascular mononuclear leukocytes) (Fig. 5B), and there was no evidence that these animals had undergone a pulmonary infection. The remaining two animals (2.4, 2.5) as well as all the mAb P7 treated animals (Fig. 5C) exhibited small consolidated areas with epithelial hyperplasia similar to the PBS control cohort, consistent with prior focal SARS-CoV-2 infection that had induced epithelial cell damage and loss, followed by regenerative processes. In the Sotrovimab control animals, histological changes were only seen in 4 / 6 lungs (Fig. 5D); these were minimal and non-specific (perivascular mononuclear leukocytes). There was no evidence that this cohort of animals had undergone a pulmonary infection. Detailed information on individual animals is provided in Table 7.

[0302] To further assess the in vivo efficacy of our antibodies, we evaluated mAb 99 and P7 at a lower dose (4 mg / kg) against live Omicron BA.5 virus in Syrian hamsters. Weight of hamsters remained stable during the experiment (data not shown) as previously reported (S. Mohandas et al., Comparative pathogenicity of BA.2.12, BA.5.2 and XBB.1 with the Delta variant in Syrian hamsters. Front Microbiol 14, 1183763 (2023)). Measuring the lung viral load seven days post-infection showed that animals treated with mAb 99 (5x103copies of N1 / pg of RNA; p = 0.0346) and P7 (3x103copies of N1 / pg of RNA; p = 0.0096) had a significant decrease compared to the PBS only control (2x104copies of N1 / pg of RNA). The control Sotrovimab was not significantly protective (7x103copies of N1 / pg of RNA; p = 0.0856; fig. 12A).

[0303] We only tested mAb 99 against Lineage A in vivo as mAb P7 was isolated after the experiment was conducted. The animals in the untreated group showed a small but not significant progressive weight loss, whereas all animals treated therapeutically showed only a small weight loss 24 h after viral challenge, and from day 2 had recovered to at least pre-challenged weights (fig. 12B). The same result was observed in animals administered with the control commercial Ronapreve (casirivimab and imdevimab) mAb cocktail. In addition to measuring hamster weight loss, we measured viral loads in the lungs on day 7. Animals treated with mAb 99 exhibited a significantly lower viral load (3x103copies of N1 / pg of RNA; p = 0.0007) than the PBS control (5x105copies of N1 / pg of RNA). Ronapreve also had a significant reduction on viral load in the lungs (9x102copies of N1 / pg of RNA; p = 0.0007; fig. 12C).

[0304] Table 7. Histological changes and viral antigen expression in the lungs of male Syrian hamsters, aged 8-10 weeks that had received PBS (cohort 1), mAb 99 (cohort 2), mAb P7 (cohort 3) or Sotrovimab (cohort4) 24 h prior to intranasal infection with 10A4 PFU SARS-CoV- 2 Delta in 200 pL and euthanised at 7 dpi.

[0305]

[0306]

[0307]

[0308]

[0309] AEC- alveolar epithelial cells (type I and II pneumocytes); BEC-bronchiolar epithelial cells; HE - histological examination of hematoxylin-eosin (HE) stained sections from the left lungs; IH - immunohistology (IH) for SARS-CoV-2 nucleoprotein; IM - intramuscular; IP -intraperitoneal; LC - lymphocytes; neg - negative (no evidence of viral antigen expression);NHAIR - No histological abnormality is recognised. NL - neutrophils; PI - pre infection; pn - pneumocytes; pv - perivascular

[0310] Discussion

[0311] The constant evolution of SARS-CoV-2 within the human population gives rise to new VOCs that can more easily escape immune reactions in the current landscape of vaccinated and infected individuals. VOCs, such as Omicron, contain mutations in key residues of spike that affect the efficacy of currently available vaccines and neutralising antibodies (S. Iketani et al., Nature 604, 553-556 (2022); W. Dejnirattisai et al., Cell 185, 467-484.e415 (2022)). Here, we showed that consecutive immunisations with Lineage-A spike in cows, generated a strong and broad immune response against all SARS-CoV-2 variants, as well as against SARS- CoV-1. A recently published study confirms our findings of generation of broad potent bovine mAbs, showing that cow immunisations with the ancestral Lineage-A spike can elicit broad antibody responses, which neutralise contemporary SARS-CoV-2 variants and / or other sarbecoviruses (R. Huang et al, Supra). The strongest and most broad immune response was observed in the final timepoint D242, which was 73 days after the previous bleed on D169 (Fig. 1A and B), consistent with previous studies showing that longer intervals between later boosts induce strong and broad immune responses (D. Sok et al., Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows. Nature, 548, 108-111 (2017); E. A. Lefevre, B. V. Carr, H. Prentice, B. Charleston, A quantitative assessment of primary and secondary immune responses in cattle using a B cell ELISPOT assay. Vet Res 40, 3 (2009)). We employed two different methods to retrieve SARS-CoV-2 spike-specific antibodies from bovine PBMCs: i) single spike-specific B-cell sorting in 96-well plates, and ii) bulk sorting of spike-specific B-cells and phage display. Both approaches have been used by previous studies to retrieve antibodies from cow PBMCs (D. Sok et al supra, R. Huang et al., The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2. Proceedings of the National Academy of Sciences 120, e2303455120 (2023)). Our data showed that, within a panel of 33 antibodies, 21 bound to the SARS-CoV-2 spike, of which 5 recognised the RBD (Fig. 1C). The RBD-binding mAbs P2, P7 (both ULs), and 99 (non-UL) showed great breadth and binding capacity against SARS-CoV-2 VOCs. mAbs 99, 103, and 101 competed with human ACE2 for RBD binding (Fig. 1 D) suggesting neutralisation is achieved without complete obstruction of the ACE2 RBD interaction, similar to mAbs S2H97 and B9-scFv (T. N. Starr et al., Nature 597, 97-102 (2021); M. J. Burke et al., Journal of Biological Chemistry 298, (2022)). Individual RBD-targeting mAbs, such as Regdanvimab andBamlanivimab (which compete with ACE2), and Sotrovimab, S2X259, and Bebtelovimab (which do not compete with ACE2) Licensed therapeutic mAbs targeting RBD, such as Regdanvimab and Bamlanivimab, are ACE2- competitive mAbs, while Sotrovimab, S2X259, and Bebtelovimab are not, as well as mAb cocktails such as Evusheld (cilgavimab + tixagevimab) and Ronapreve (casirivimab + imdevimab) have been used as therapeutics for SARS-CoV-2 (M. Cox et al., SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies. Nature Reviews Microbiology 21, 112-124 (2023); S. Drozdzal et al., An update on drugs with therapeutic potential for SARS-CoV-2 (COVID-19) treatment. Drug Resistance Updates 59, 100794 (2021)), although their lack of effectiveness against some VOCs has led to their removal from clinical use. This highlights the need to isolate alternative, next-generation mAbs to overcome the threat of escape.

[0312] Further characterisation of the 21 spike-binding mAbs in single-point neutralisation assays against pseudotyped SARS-CoV-2 spikes revealed the best and most broadly neutralising mAbs. mAbs P2, P7, 118, 105 (ULs) and 99 (non-UL) were selected and further characterised because they could neutralise at least 5 out of 6 variants, with a minimum of 75% efficacy (Fig. 2A). Comparison of the in vitro IC50 values of our mAbs to the licenced therapeutic mAbs showed that mAbs P2 and 99 had similar neutralisation potencies to Amubarvimab, Romlusevimab, and Sotrovimab for variants Alpha, Beta, and Delta. Licenced antibodies Sotrovimab and Romlusevimab showed a 3-fold reduced potency against the Omicron (B.1.1.529) variant, whereas most of the other therapeutic antibodies except for Bebtelovimab, showed great reductions in neutralisation against this variant (M. Cox et al., supra, E. Cameroni etal., Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature 602, 664-670 (2022)). In contrast, our antibodies were still in the range of IC50 10-50 ng / mL against Omicron. The mAbs P7 and 99 were able to potently neutralise more contemporary variants such as Omicron BA.5 and XBB (Fig. 2B and C). The most broadly neutralising mAb was P7, which neutralised SARS-CoV-1 and bound RBDs across several sarbecovirus clades, suggesting it targets a conserved epitope within sarbecovirus RBDs (Fig. 3).

[0313] In addition to SARS-CoV-1 cross-neutralisation, mAb P7 also showed cross- reactivity against RBD present in representative animal sarbecoviruses drawn from clades 1a, 1b, 2 and-3. Pan- coronavirus cross-recognition has been observed for some monoclonals targeting the NTD of spike, but the reported affinity and potency of these antibodies is low (N. K. Hurlburt etal., Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit. Commun Biol 5, 342 (2022); J. Hurtado et al., Deep repertoire mininguncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes. Cell Rep 43, 114307 (2024)). Several broadly reactive pan-sarbecovirus RBD-targetting murine and human mAbs have been reported, and these include S309 (Sotrovimab), 2-36, M2-7, M8a-31, M8a-34, CC25.54 and CC25.36 (C. Fan et al., Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes. Immunity 55, 2419-2435. e2410 (2022)., D. Pinto et al., Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020); P. Wang et al., A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses. Emerg Microbes Infect 11, 147-157 (2022); C. Liu et al., Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses. Cell Rep 43, 114235 (2024); W T. He et al., Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS- like viruses. Nat Immunol 23, 960-970 (2022)).

[0314] A common feature of these human RBD-targeting mAbs with broad reactivity is their relatively extended CDRH3 (>20 amino acids). Given P7's ultralong CDRH3, it is possible that possession of a CDRH3 with increased length gives favourable cross- reactivity, by enabling engagement with highly conserved epitopes that are less accessible to mAbs with shorter CDRH3s.

[0315] The efficacy of the candidate mAbs in vivo, showed a level of protection that was at least equivalent to commercial mAb therapeutics; Ronapreve (a cocktail of casirivimab and imdevimab, both lgG1) against Lineage A and Sotrovimab against Delta and Omicron BA.5 (Fig. 4). Of note is that the antibodies presented here do not have Fc modifications to improve their in vivo half-life unlike Sotrovimab (LS mutation (Met428Leu / Asn434Ser)) (D. Focosi, A. Casadevall, M. Franchini, F. Maggi, Sotrovimab: A Review of Its Efficacy against SARS-CoV-2 Variants. Viruses 16, (2024)), suggesting that they may be even more efficacious with the same modifications. Our data showed that, although the cows were immunised with Lineage-A spike, we discovered antibodies that could bind several animal sarbecovirus RBDs. Assessment of the binding capability of our RBD-binding mAbs against a panel of sarbecovirus RBDs showed that the most broadly reactive mAb was P7 (23 of 23), followed by 99 (8 of 23), P2 (7 of 23), 103 (4 of 23) and 101 (3 of 23) (Fig. 2). Overall, our study not only demonstrates the isolation of neutralising mAbs from immunised cows, but also highlights the discovery of two new broadly neutralising mAbs. These antibodies show great potential for identifying conserved epitopes, and as therapeutic candidates against SARS-CoV-2, with P7, in particular, standing out as a pan-sarbecov / s-reactive antibody that could serve to inform vaccine design, as a diagnostic tool, and as a pandemic response therapeutic.Materials and methods

[0316] Immunisations

[0317] Two Holstein bullocks were immunised each with 250 g of the S1 subunit of SARS-CoV-2 spike (REC31806, Native Antigen Company) mixed with Titremax Gold Adjuvant (Sigma Aldrich). A booster immunisation was performed with 150 pg of the same protein / adjuvant combination 21 and 42 days after the prime immunisation. Another booster immunisation was performed 161 days post-prime, with 250 pg of SARS-CoV-2 spike ectodomain (S1 and partial S2; aa 1-1211 , SRAS furin cleavage site, transmembrane domain and intravirion part replaced with a glycine-serine linker; REC31868, Native Antigen Company) mixed with Titremax Gold Adjuvant (Sigma Aldrich). A final boost was performed on day 235 with 200 pg of SARS-CoV-2 trimeric spike (aa 1-1208, GSAS furin cleavage site, soluble 2P (aa 986-987), purity and size verified by manufacturer; REC31871, Native Antigen Company) mixed with the same adjuvant. Blood (50 mL) was collected on days 25, 46, 50, 165, 169, 239 and 242 - approximately 4- and 8-days post immunisation. Also, blood was collected on day 0 as a control.

[0318] Blood processing

[0319] Peripheral blood mononuclear cells (PBMC) and plasma were harvested after processing the blood samples with Histopaque-1077 (Sigma-Aldrich) and SepMate PBMC isolation tubes (Stemcell Technologies). PBMCs and plasma were stored in LN2 and -80 °C, respectively. The plasma samples were heat inactivated at 56 °C for 40 min before use in assays.

[0320] Flow cytometry sorting

[0321] Twenty million (20 x 106) PBMCs were defrosted for each bullock and incubated O / N at 37 °C in 5% CO2 in Gibco™ RPMI 1640 Medium supplemented with 10% heat inactivated fetal bovine serum (FBS). The cells were washed with 3% bovine serum albumin (BSA, Sigma) dissolved in phosphate-buffered saline (PBS) with the addition of 2 mM ethylenediaminetetraacetic acid (EDTA, Sigma). After centrifugating at 300g for 10 minutes and then washing again, the cells were stained with biotinylated Lineage A trimeric spike (The Native Antigen Company) for 30 min at 4 °C followed by washing, centrifugating and staining with R-PE and APC conjugated streptavidin (Thermo Fisher Scientific) for 30 min at 4 °C. After the final wash, the cells were stained with Annexin-V FITC and propidium iodide (PI, Thermo Fisher Scientific) for 20 min at 4 °C. Using an Astrios cell sorter (Beckman Coulter), cells were gated using FSC / SSC plots and aggregates excluded by SSC pulse width. The lymphocyte gate was identified based on forward and side scatter. Doublets were then excluded, and Annexin-V FITC and PI cell dyes were used for live-dead discrimination. R-PE+and APC+spike-specific labelled B-cells were gated as our primarypopulation of interest and were sorted into 96-well plates containing 1 mg / mL BSA (Thermo Fisher Scientific) and were frozen immediately at -80 °C. In addition to single cells, a bulk sort of live spike-specific B-cells was also performed for each bullock, which was then used for phage display.

[0322] Retrieval of antibody heavy and light chains and cloning

[0323] After the sorted single cells were snap frozen, cDNA was synthesised using RNA to cDNA EcoDry Premix Random Hexamers (Takara Bio) following the manufacturer's instructions. Full-nested PCR was performed to retrieve the heavy and light chain for each B-cell using HotStartTag DNA polymerase (Qiagen) following the manufacturer's conditions. The first round was a multiplex targeting the IgG heavy chains and the lambda light chains (I g L) . The second round PCRs were targeting IgG and IgL separately. Each IgH PCR product was cloned into an lgG1 cassette in pcDNA3.1, which was comprised of human constant region and bovine variable regions. Similarly, the IgL PCR products were cloned into a human IgL cassette containing bovine variable regions. The cloning was performed using In-Fusion cloning (Takara Bio) following the manufacturer's protocol. The Fab fragment versions of the cloned antibodies were generating by removing their Fc region using a Q5 Site Directed Mutagenesis kit (New England Biolabs).

[0324] Generation of Phage Libraries from bulk sorted anti-specific B cells

[0325] RNA was extracted from bulk sorted SARS-CoV-2 spike reactive B-cells using an RNA extraction kit (Qiagen), according to manufacturer's instructions. Purified RNA was used as the template for

[0326] cDNA synthesis using EcoDry Premix random hexamers (Takara Bio). Heavy chain variable regions of VH1-7 family were amplified from the cDNA by PCR using VH family specific primers. Retrieved VH1-7 amplicons were subcloned into a linearised phagemid pFab-VL vector by Gibson assembly (New England BioLabs) according to manufacturer's instructions. The pFAb- VL vector encoded a bovine CL and CH1 framework and a previously described VL1-47 light chain variable region (25). The bovine Fab was placed upstream of a truncated phage gene 3 sequence to allow incorporation of Fab into phage particles on the p3 minor protein. Products of the Gibson assembly were electroporated in TG1 E. coli cells (Lucigen) and transformed cells were grown on 2XYT supplemented with 2% glucose and 100 pg / mL ampicillin (2XYT GA) agar plates and incubated at 37 °C overnight.

[0327] Phagemid libraries were rescued by harvesting colonies from each culture plates into 25 mL2XYT GA media and grown until log phase when M13K07 helper phage (New England BioLabs) was added at an MOI 1.25. Cultures were incubated with helper phage for 45 min at 37 °C and then pelleted by centrifugation at 3000 x g for 10 min. Supernatant was discard and pelleted cells were resuspended in glucose free media and grown for 18 h at 30 °C. Phage particles were harvested from infected cultures by removal of bacterial cells by centrifugation followed addition of PEG precipitation buffer (20 % PEG8000, 2.5 M NaCI). Culture supernatants were incubated in PEG buffer overnight at 4 °C and precipitates were pelleted by centrifugation at 4000 x g for 10 min followed by resuspension in PBS. Phage particles were quantified by infecting TG1 E. coli followed by titration and inoculation of 2XYT GA agar plates to calculate colony forming units per pL.

[0328]

[0329] Phage libraries were normalised to a concentration of 1 x1011cfu / well and added to a 96 well maxisorp plate (Thermo Fisher Scientific) that had been coated with trimeric SARS-CoV-2 spike from either Lineage A (first round) or BA5 (second round) variants, at a concentration of 5 pg / mL and blocked with 3% BSA for the first round, and 3% Gelatin from cold water fish skin. Phage particles were incubated on the plate for 1 hr at room temperature with gentle agitation followed by 20 washes with PBST with increasing concentrations of Tween20 from 0.1 % to 1.0%. Bound phage particles were eluted from the plate by the addition of acidic glycine buffer (0.2 M glycine, p / 72.2) and incubated for 10 minutes followed by neutralisation with 1 M Trizma hydrochloride (Tris HCL, p / 79.1). Eluted phage was added to 600 pL of TG1 cells that were at log-phase and cells were cultured for 1 hour then plated on 2XYT GA agar. Colonies were phage rescued and titrated 24 hrs later as previously described.

[0330] Mammalian cell culture

[0331] HEK-293T and Vero-E6 adherent cell lines were maintained in DMEM supplemented with 10 % FBS (Sigma-Aldrich) and 1 % non-essential amino acids (Gibco) and grown at 37 °C with 5% CO2. The HEK derived suspension cell line, Expi293 (Thermo-Scientific) was maintained in Expi293 cell media (Thermo-Scientific) and propagated at 37 °C, 8% CO2 with agitation at 125 rpm.

[0332] Expression of recombinant mAbs

[0333] Small scale expression of the mAb panel was initially performed using transient transfection of HEK-293T cells. In brief, 4 pg of heavy chain-encoding plasmid and 4 pg of light chainencoding plasmid were prepared for transfection by mixing with 16 pg P3000 reagent (Thermo Fisher Scientific), 24 pg Lipofectamine reagent (Thermo Fisher Scientific) and Opti-MEM (Gibco), made up to a final volume of 500 pL. This solution was incubated for 20 minat room temperature and was added to a 10 cm Primaria™ coated dishes (Corning) that had been seeded with 3x106HEK- 293T cells 24 hrs prior to transfection. The cells were incubated for 6 hrs and the media was replaced with 10 mL of fresh DMEM. Transfected cultures were grown for 72 hrs and the supernatant was harvested and passed through a 0.22 pM syringe filter (Sartorious).

[0334] For larger scale recombinant mAb production, the Expi293 expression system (Thermo Fisher Scientific) was used. Expi293 cells cultures were seeded at 2.5 x106cell / mL 24 hrs prior to transfection and incubated at 37°C, 8% CO2 with shaking at 125 rpm. Cell densities were adjusted to 3 x106cells / mL and transfected with heavy and light chain encoding plasmids at a ratio of 1:2, respectively, with the total amount of plasmid equivalent to a final concentration of 1 pg / mL of DNA in the transfected culture. Cultures were harvested 7 days post-transfection and centrifuged at 300 x g for 20 min to remove cells followed by centrifugation at 4500 x g for 1 hr to remove subcellular debris.

[0335] Purification of recombinant mAbs

[0336] Supernatants harvested from mAb expressing Expi-293 cultures were diluted at a 1:1 ratio with binding buffer (100 mM sodium phosphate, p / 77.0) and passed through a 0.22 pm filter (StarLab). Recombinant mAb was purified from samples using an AKTA start system (Cytiva) equipped with a 1 mL HiTrap™ Protein G HP column (Cytiva). All steps were performed at a flow rate of 2 mL / min. In brief, the protein G column was equilibrated with 10 column volumes (CVs) prior to addition of the sample. Sample was applied to the column followed by a washing step with 15 CVs of binding buffer. Captured mAb was eluted from the column using 15 CVs of elution buffer (200 mM glycine, p / 72.7). Eluted sample was collected as 1 mL fractions thatwere mixed with 200 pL of neutralisation buffer(1 M Tris, p / 79.0). Fractions that contained the elution peak were pooled and concentrated using an Ultra-4 centrifugal filter device (Amicon) with a molecularweight cut- off of 100 KDa. Removal of residual glycine was achieved by buffer exchange using a zeba column (Thermo fisher scientific) equilibrated with PBS. Purified mAbs were then stored at -20 °C for future use.

[0337]

[0338] Recombinant mAbs were quantified using an in-house IgG ELISA. In brief, 96 well maxisorp ELISA plates (Thermo Fisher Scientific) were coated with anti-human IgG heavy chain antibody (12136-1 ML, Sigma-Aldrich) at a concentration of 10 pg / mL, diluted in carbonate-bicarbonate buffer, at a volume of 50 pL / well. Following overnight incubation at 4 °C, coating buffer was removed, and plates were blocked with Phosphate-Buffered Saline with 0.1% Tween 20 (PBST) and 5% skimmed milk for 2 hrs at room temperature then washed once with PBST buffer. Samples were serially diluted, alongside a standard of recombinant human lgG1 Lambda antibody (Sigma-Aldrich) that was used to generate a standard curve starting from 20 pg / mL and diluted 2-fold for 16 points. Standards and samples were incubated on the plate for 90 min at room temperature and were removed followed by three washes with PBST. Captured IgG was detected by a 1 hr incubation with an anti-human Lambda chain Alkaline phosphatase secondary antibody at a 1:2000 dilution in PBST. Following removal of the secondary antibody, ELISA plates were washed a further three times with PBST and detection of bound antibody complexes were measured by the addition of para-Nitrophenylphosphate (pNPP) (Sigma-Aldrich) colorimetric substrate with absorbance read at 405 nM. IgG concentration of samples was interpolated from the standard curve.

[0339]

[0340] A panel of sarbecovirus RBDs similar to those described in (C. Fan et al., Immunity 55, 2419-2435. e2410 (2022).) were expressed in HEK 293T cells. Briefly, 4 pg of plasmid encoding a His-tagged RBD was mixed with p3000 and lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer's instructions, and transfected into a 10 cm dish of 3 x 106HEK 293T cells. Supernatant was harvested 72 hrs post transfection and soluble RBD proteins were purified using nickel-affinity purification. All centrifugation steps were carried out at 1000 x g for 1 minute. In brief, supernatant was incubated with 200 pL of Ni- NTA His bind resin (Merck) and incubated for 1 hr at room temperature. A resin solution was added to a 10 KDa Amicon Ultra-0.5 device (Merck) and supernatant removed by centrifugation. Resin was washed in 50 mM phosphate buffer (p / 78.0, 10 mM Imidazole) and flow through was discarded. The sample was added to the exchange device and incubated for 1 hour at room temperature with gentle agitation. Unbound protein was removed by centrifugation and flow through discarded. The device was subsequently washed with wash buffer, centrifuged and flowthrough discarded. Bound protein was eluted from the resin by addition of elution buffer (50 mM phosphate buffer, p / 78.0, 0.3NaCI, 25 mM Imidazole) and incubated for 5 minutes at room temperature. The eluted protein was collected by centrifugation at 4000 x g for 15 minutes. Eluted protein was validated by SUBPAGE and silver staining.

[0341] Binding ELISASARS-CoV-2 antigens were coated onto 96 well maxisorp plates (Thermo-Scientific) at a concentration of 500 ng / mL, and 5 pg / ml for the panel of 23 sarbecovirus RBDs, and incubated at 4 °C overnight. Plates were blocked with 5% skimmed milk PBST for 2 hrs. Recombinant antibodies were serially diluted 3- fold with a starting concentration of 2.5 pg / mL in duplicate and incubated on the ELISA plate for 1 hr. The wells were washed three times with PBST and anti- human IgG-Fc specific alkaline phosphatase (A3187, Sigma) was added at a 1:2000 dilution followed by 1 hr incubation. The assay was developed using SIGMAFAST™ p-Nitrophenyl phosphate (pNPP) Tablets (Sigma) according to manufacturer's procedure and absorbance read at an optical density of 405 nm, using a Fluorostar Omega microplate reader (BMG Labtech, Aylesbury, UK), in 5-minute intervals until saturation was reached. Analyses of results were carried out using GraphPad Prism version 10 for macOS (Graphpad Software, San Diego, USA). Analyses as proportion of binding (%) relative to the maximum binding (2.5 pg / mL antibody concentration) and minimum binding negative control)). The EC50 was calculated using a non- linear analysis model.

[0342] ACE2 inhibition ELISA was performed similar to the binding ELISA, except recombinant antibodies were serially diluted four-fold in ACE2 / PBST starting at a concentration of 2.5 pg / mL in duplicate and incubated on the ELISA plate for 1 hr. The wells were washed three times with PBST and Monoclonal Anti- Goat / sheep Alkaline Phosphatase antibody produced in mouse (Sigma: A8062) was added at a 1:2000 dilution followed by 1 hour incubation. The assay was developed and analysed as previously mentioned.

[0343] Retroviral pseudotype particle neutralisation assay

[0344] Neutralising activities of bovine sera and recombinant monoclonal antibodies were assessed using a SARS-CoV-2 retroviral pseudotype (SARS-CoV-2 pp) assay, as described previously (R. A. Urbanowicz etal., Science Translational Medicine 13, eabj0847 (2021). In brief, 1.5 x106HEK-293T cells were seeded in a 10 cm Primaria coated dish (Corning) in 10 mL of DMEM, 24 hrs prior to transfections. The following day, 2 pg of MLV gag-pol packaging plasmid (phCMV-5349), 2 pg MLV- luciferase plasmid (pTG126) and 2 pg of pcDNA3.1 plasmid (Invitrogen) encoding SARS-CoV-2 spike proteins, were mixed with 24 pL polyethylenimine (Polysciences) diluted in Opti-MEM (Gibco) and incubated for 1 hr a room temperature. Plasmids were added to cells and incubated for 6 hrs at 37 °C and then removed and replaced with 10 mL of DMEM. Supernatants of transfected cell cultures were harvested 72 hrs post-transfection and passed through a 0.45 pM syringe filter (Sartorius)and stored at 4 °C and used within 1 week of harvesting. For neutralisation assays, 270 pL of SARS-CoV-2 pps were mixed with either 30 pL of diluted recombinant antibody or diluted heat inactivated Bovine plasma and incubated at room temperature for 1 hr. SARS-COV-2 pps were then added in triplicate to a white 96 well plate (Corning) that had been seeded with 2 x104Vero-E6 cells 24 hrs prior. Vero-E6 cells were incubated with SARS-CoV-2 pps for 4 hrs at 37 °C followed by removal of pseudotyped viruses and addition of 200 pL of DMEM. Vero-E6 cells were incubated for a further 72 hrs at 37°C, 5 % CO2. Luciferase activity was measured using the Promega luciferase assay system, cell supernatant was removed from the 96 well plate and 50 pL of cell lysis buffer was added per well and incubated a room temperature for 30 min with agitation followed by addition of 50 pL luciferase substrate solution per well. Luminescence was measured using a FLUOstar Omega plate reader (BMG Labtech). Analyses of results were carried out using GraphPad Prism version 10 for macOS (Graphpad Software, San Diego, USA). Luminescence was normalised to positive and negative controls, calculated as proportion of neutralisation relative to the maximum infectivity (positive control) and minimum infectivity (delta Envelope pseudovirus particles). The IC50 for curves was calculated on GraphPad Prism by transforming the data and using a non-linear analysis model.

[0345] Authentic virus neutralisation assay

[0346] Authentic SARS-CoV-2 neutralisation assays were performed as previously described (56) except that 3.95 x103TCID50 of virus was added to each well and each antibody was diluted from 5 pg / mL down a 4-fold dilution series in triplicate. For this assay, antibodies P7 and 99 were tested against SARS-CoV-2 variants Lineage A, Alpha, Beta, Delta and Omicron. In vivo infection studies

[0347] Animal work was approved by the local University of Liverpool Animal Welfare and Ethical Review Body and performed under UK Home Office Project Licence PP4715265. Male golden Syrian hamsters (Mesocricetus auratus) aged between 8 and 10 weeks old, weighing 90-100 g were purchased from Janvier Labs (France). Animals were maintained under SPF barrier conditions in individually ventilated cages. For virus infections the following strains were used: Liverpool strain, a PANGO lineage B strain of SARS-CoV-2 (hCoV- 2 / human / Liverpool / REMRQ0001 / 2020) was cultured from a nasopharyngeal swab from a patient, was passaged in Vero E6 cells (Patterson et al., 2020). The B.1.617.2 (Delta variant) hCoV- 19 / England / SHEF-10E8F3B / 2021 (GISAID accession number EPI_ISL_1731019) and BA.5 was provided by Prof. Wendy Barclay, Imperial College London, London, UK through the Genotype- to-Phenotype National Virology Consortium(G2P-LIK). All virus stocks had been checked for deletions in the mapped reads and the stocks confirmed to not contain any deletions.

[0348] Animals were randomly assigned into multiple cohorts of 6. For SARS-CoV-2 infection, hamsters were anaesthetised lightly with 5% isoflurane and inoculated intranasally with 100 pl containing 104PFU of SARS-CoV-2 Delta variant and 105PFU of Liverpool and BA.5 variants in PBS. At day -1 pre-challenge (pc), for the Delta variant infection, hamsters were treated with 10 mg / kg of mAbs 99 and P7 via the intraperitoneal route (l / P) in 200 pl PBS. Control hamsters (n = 6) received either no treatment (negative control) or Sotrovimab at 10 mg / kg body weight as a positive control. For BA.5, hamsters were treated at day -1 pc with 4 mg / kg of mAbs 99 and P7 via the intraperitoneal route (l / P) in 200 pl PBS. Control hamsters (n = 6) received either no treatment (negative control) or Sotrovimab at 4mg / kg body weight as a positive control. For the Liverpool variant, hamsters were treated at day -1 pc with 4 mg / kg of mAb 99 via the intraperitoneal route (l / P) in 200 pl PBS. Control hamsters (n = 6) received either no treatment (negative control) or Ronapreve (REGN-COV2; 10 mg / kg body weight l / P) as a positive control.

[0349] Hamsters were monitored daily for weight loss. Oropharyngeal (throat) swabs were obtained on days 3 and 5 post infection. Animals were briefly anaesthetised with 5% isoflurane for the collection of these samples. On day 7, all hamsters were euthanised by an overdose of anaesthetic (sodium pentobarbitone) via the intraperitoneal route. At necropsy, nasal turbinates and tissue samples (right lung) were collected and stored frozen at -80 °C for viral RNA measurement. From animals that had been infected with SARS-CoV-2 Delta and were treated with mAb 99, mAb P7 and Sotrovimab, the left lung was fixed in 10% buffered formalin for histological and immunohistological examination.

[0350] RNA Extraction and qRT-PCR

[0351] RNA was isolated from oropharyngeal swabs and tissue samples (lung and nasal turbinates). Right upper lung lobe and nasal turbinates were homogenised in 1 ml of Trizol reagent (Thermofisher) using a QT tissue lyser and stainless-steel beads (Qiagen) at 50 oscillation for 5 minutes whereas swabs samples were inactivated in 1:3 ratio of Trizol LS (Thermofisher). The homogenates were clarified by centrifugation at 12,000xg for 5 min before full RNA extraction was carried out according to manufacturer's instructions. RNA was quantified and quality assessed using a Nanodrop (Thermofisher) before a total of1pg was DNase treated using the TURBO DNA-free™ Kit (Thermofisher) as per manufacturer's instructions.

[0352] Viral load was quantified using a 1 step RT-qPCR kit (Promega). For quantification of SARS-COV-2 the nCOV_N1 primer / probe mix from the SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay (IDT) were utilised and murine 18s was used as a housekeeping gene to normalise the qPCR. The 18s standard was generated by the amplification of a fragment of the murine 18S cDNA using the primers F: ACCTGGTTGATCCTGCCAGGTAGC and R: AGO CAT TCG CAG TTT TGT AC. Likewise, SARS-COV2 genomic N1 standard was generated by PCR using the qPCR primers. cDNA was generated using 1 step qRT-PCR kit (Promega) as per manufacturer's instructions. Both PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and serially diluted 10-fold from 1010to 104copies / reaction to form the standard curve.

[0353] Histological and immunohistological examination

[0354] After fixation in 10% buffered formalin for 48 h, the left lungs of all hamsters were transferred into 70% ethanol until further processing. The lung was halved by a longitudinal section and routinely paraffin wax embedded. Consecutive sections (3-4 pm) were prepared and stained with hematoxylin eosin (HE) for histological examination or subjected to immunohistological staining. Immunohistology was performed to detect SARS-CoV-2 antigen, using the horseradish peroxidase (HRP) method and the following primary antibody: rabbit anti-SARS-CoV nucleocapsid protein (Rockland, 200-402-A50) as previously described (J. Huo et al. , A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nat Commun 12, 5469 (2021)).

[0355] Protein expression and purification

[0356] The HexaPro (6P) version of SARS-CoV-2 Lineage A spike trimer (C.-L. Hsieh et al., Structure- based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369, 1501-1505 (2020)), SARS-CoV-2 Lineage A RBD, Fabs from mAbs P2, P7, 105, 115, and 118, and 99 IgG were expressed in Expi293 cells by transient transfection as described (C. O. Barnes et al supra). Proteins were purified following previously-established protocols (C. Fan et al., supra). Briefly, His-tagged spike trimer was purified from Expi293 cell supernatant using a Ni-NTA column followed by size exclusion chromatography (SEC) using a Superose 6 10 / 300 column (Cytiva). Peak fractions were collected and concentrated to ~6 mg / mL and flash frozen in 30 pL aliquots and stored in -80°C until use. His-tagged SARS-CoV-2 RBD and P2, P7, 10,5 115 and 118 Fabs were purified from cell supernatants using a Ni-NTA column, and eluents from NiNTA purifications were subjected to SEC using a Superdex 20010 / 300 Increase column (Cytiva). For making RBD-Fab complexes, both proteins were coexpressed during a transient transfection in Expi293 cells. Peak fractions from SEC were pooled and concentrated to ~25 mg / ml and screened for crystallization. 99 IgG was purified by a MabSelect SuRe column followed by SEC purification using a Superdex 200 10 / 300 Increase column (Cytiva). 99 Fab was generated by papain digestion from purified 99 IgG as described (K. Vanshylla et al., supra).

[0357] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0358] All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0359] Each feature disclosed in this specification (including any accompanying claims, abstract, and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0360] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any novel one or any novel combination of the steps of any method or process so disclosed.

Claims

Claims1. An antibody molecule or an antigen binding fragment, wherein the antibody molecule or antigen binding fragment:i) comprises a bovine CDR-H3 or a fragment thereof which has a sequence selected from the list consisting of SEQ ID NO. 5, 3, 8, 10, 12, 15, 18, 20, 23, 26, 29, 32, 35, 37, 40, 43, 45, 48, 51 or 54 of Table 1;ii) reduces or inhibits binding of the ACE2 receptor to a SARS related coronavirus spike protein; and / oriii) binds to i) the S1 protein (e.g. amino acids 1-674 of the Lineage A S protein); ii) the S protein ectodomain (e.g. amino acids 1-1211 of the Lineage AS protein); iii) the full length S protein (e.g. amino acids 1-1208 of the Lineage A S protein); and / or iv) the RBD (e.g amino acids 319-541 of the Lineage AS protein); and / or the corresponding sequence in a SAR-CoV-2 variant; or a fragment thereof which is capable of eliciting a SARS related coronavirus specific immune response 2. An antibody molecule or antigen binding fragment according to claim 1, wherein the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 which has a sequence of SEQ ID. NO. 5.

3. An antibody molecule or an antigen binding fragment according to claim 1 or claim 2, comprising a VHCDR1 which has the sequence of SEQ ID NO. 1, a VHCDR2 which has the sequence of SEQ ID NO. 4 and a VHCDR3 which has the sequence of SEQ ID NO. 5.

4. An antibody molecule or an antigen binding fragment according to claim 1, wherein the antibody molecule or antigen binding fragment comprises a bovine CDR-H3 or a fragment thereof which has a sequence of SEQ ID. NO. 3, SEQ ID. NO. 8, SEQ ID. NO. 26, or SEQ ID. NO. 29.

5. An antibody molecule or antigen binding fragment according to any one of claims 1 to 4, wherein the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 1, 6, 13, 16, 21, 24, 27, 30, 33, 38, 41, 46, 49 and 52 of Table 1; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of selected from the list consisting of SEQ ID NO. 2, 4, 7, 9, 11, 14, 17, 19, 22, 25, 28, 31, 3436, 39, 42, 44, 47, 50 and 53 of Table 1.

6. An antibody molecule or antigen binding fragment, wherein the antibody molecule or antigen binding fragment comprises a heavy chain variable region (VH) selected from the list consisting of SEQ ID NO. 96-115 of Table 3, suitably SEQ ID NO. 97.

7. An antibody molecule or antigen binding fragment according to any one of claims 1 to 6, wherein the antibody molecule or antigen binding fragment neutralises at least 65% of the infectivity of SARS-CoV-2 Lineage A, SARS-CoV-2 Alpha, Beta, Gamma, Delta and / or Omicron (B.1.1.529) variants.

8. An antibody molecule or antigen binding fragment according to any one of claims claim 1 to 7, wherein the antibody molecule or antigen binding fragment binds to an RBD epitope in at least 3 SARS related coronaviruses selected from the group consisting of RMYN02, Yun11, RS4081, RF1, KHOSTA2, BTKY72, BM4831, SCH014, WIV1, LYRA3, CO28, RSSTT200, PANG17, RATG13, BA5.58, BA2.75.58, BQ1.1, BA2.75.2, XBB, BA2, and FK.

9. An antibody molecule or antigen binding fragment according to any one of claims claims 1 to 8, wherein the antibody molecule or antigen binding fragment inhibits binding of a SARS related coronavirus to an ACE2 receptor.

10. An antibody molecule or antigen binding fragment comprising a knob fragment of a bovine ultra-long CDR-HR which has a sequence selected from the list consisting of SEQ ID NO. 3, 5, 10, 12, 23, 37 or 40 of Table 1.

11. An antibody molecule or antigen binding fragment according to any one of the preceding claims, comprising a light chain variable region (VL) or fragment thereof, wherein the light chain variable region is selected from:i) a generic variable light chain, preferably a VL of an IgL or a fragment thereof; ii) a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of selected from the list consisting of SEQ ID NO. 57, 60, 63, 64, 65, 68, 71, 74, 77, 79, 81, 83, 86, 89, 92 and 95 of Table 2iii) a light chain variable region comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence selected from the list consisting of SEQ ID NO. 55, 58, 61, 66, 69, 72, 75, 78, 80, 82, 84, 87, 90, and 93 of Table 2, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence selected from the list consisting of SEQ ID NO. 56, 59, 62, 67, 70, 73, 76, 85, 88, 91 and 94 of Table 2 and / or a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of selected from the list consisting of SEQ ID NO. 57, 60, 63, 64, 65, 68, 71, 74, 77, 79, 81, 83, 86, 89, 92 and 95 of Table 2; oriv) a combination of light chain complementarity determining region 1 (VLCDR1), light chain complementarity determining region 2 (VLCDR2) and light chain complementarity determining region 3 (VLCDR3) selected from the combinationsof SEQ ID Nos. 55, 56 and 57 (114); 58, 59 and 60 (113); 61, 62 and 63 (106, 68 and 115); 61, 62 and 64 (118); 61, 62 and 65 (105); 66, 67 and 68 (103); 69, 70 and 71 (101); 72, 73 and 74 (99); 75, 76 and 77 (73); 78, 73 and 79 (72); 80, 62 and 81 (66); 82, 76 and 83 (63); 84, 85 and 86 (60); 87, 88 and 89 (58); 90, 91 and 92 (54); or 93, 94 and 95 (53).v) a light chain variable region selected from the list consisting of SEQ ID NO. 116- 133 of Table 4, or a fragment thereof.

12. An antibody molecule or antigen binding garment according to claim 2 or 3, or SEQ ID NO. 97 of claim 6, wherein the antibody molecule or antigen binding garment comprises a light chain variable region (VL) or fragment thereof, wherein the light chain variable region is of an IgL.

13. An antibody molecule or antigen binding fragment according to any one of the preceding claims, wherein the antibody molecule or antigen binding fragment is isolated or recombinant.

14. A fusion protein comprising an antigen binding fragment as defined in any one of the preceding claims, and a further polypeptide.

15. A nucleic acid molecule encoding an antibody molecule or antigen binding fragment according to any one of the preceding claims.

16. An expression vector comprising a nucleic acid molecule according to claim 15.

17. A host cell comprising a nucleic acid molecule and / or expression vector according to claim 15 or 16.

18. A pharmaceutical composition comprising an antibody molecule or antigen binding fragment, a fusion protein, a nucleic acid molecule, an expression vector, and / or a host cell according to any one of the preceding claims; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

19. A method of producing an antibody molecule or antigen binding fragment, a fusion protein or a nucleic acid molecule according to any one of claims 1 to 14, the method comprising culturing a host cell according to claim 17 under conditions suitable for gene expression.

20. A method of producing an antibody molecule or antigen binding fragment or a fusion protein according to any one of claims 1 to 14, comprising i) a step of immunizing a host animal with an amino acid sequence comprising amino acids 1-319 or 541- 1208 and / or 319-541 of the Lineage A spike protein of SARS-CoV-2 as an antigen, or a fragment thereof which is capable of eliciting an SARS related coronavirus immune response; and ii) a step of producing a polyclonal or monoclonal antibody.

21. A method of treating a subject, wherein the method comprises administering to the subject an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any one of claims 1 to 17.

22. An antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any one of claims 1 to 17 or use in treating a viral infection, in a patient in need thereof.

23. A method of treating a viral infection in a subject, wherein the method comprises administering to the subject an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any one of claims 1 to 17.

24. A method according to claim 23, wherein the viral infection is a SARS-related coronavirus infection, preferably SARS-CoV-2 or a variant thereof.

25. An antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in preventing a viral infection, in a patient in need thereof.

26. A method of diagnosing a viral infection in a subject, wherein the method comprises contacting a sample obtained from the subject with an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any one of claims 1 to 17; and detecting binding of the antibody molecule or antigen binding fragment, nucleic acid, expression vector, fusion protein, host cell, and / or pharmaceutical composition to any SARS-related coronavirus, suitably SARS-CoV-2 or variant thereof, which is present in the sample.A kit for detecting, treating or preventing a viral infection, wherein the kit comprises an antibody molecule or antigen binding fragment, a nucleic acid, an expression vector, a fusion protein, a host cell, and / or a pharmaceutical composition according to any one of claims 1 to 17.

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