WO2022075921A1

WO2022075921A1 - Sars-cov-2 spike protein antigen-binding molecules

Info

Publication number: WO2022075921A1
Application number: PCT/SG2021/050603
Authority: WO
Inventors: Jerome Douglas BOYD-KIRKUP; Dipti THAKKAR; Piers INGRAM; Konrad PASZKIEWICZ; Siyu GUAN; Brendon John Hanson; En Zuo Conrad CHAN; Pei Chiew Angeline LIM; Damian O’CONNELL; Venkateshan Srirangam PRATIVADIBHAYANKARAM
Original assignee: Hummingbird Bioscience Holdings Limited; Dso National Laboratories; Agency For Science, Technology And Research
Priority date: 2020-10-08
Filing date: 2021-10-07
Publication date: 2022-04-14

Abstract

Antigen-binding molecules that bind to SARS-CoV-2 spike protein are provided, along with compositions comprising the same. Also provided are methods of medical treatment and prophylaxis, detection or diagnosis using said antigen-binding molecules.

Description

SARS-CoV-2 Spike Protein Antiaen-Bindina Molecules

This application claims priority from SG10202010008W filed 8 October 2020, from

SG10202010614V filed 26 October 2020, and from SG10202108612Y filed 6 August 2021 , the contents and elements of which are herein incorporated by reference for all purposes.

Technical Field

The present disclosure relates to the fields of molecular biology, more specifically antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis.

Background

In December 2019, a cluster of novel pneumonia cases (later named COVID19) emerged in the city of Wuhan, China, rapidly spreading through human-to-human transmission and with the potential to cause severe respiratory distress and significant mortality [1 , 2], High throughput sequencing of patient-derived samples indicated that the etiological agent was a novel betacoronavirus with 79.6% sequence homology to SARS-CoV and likely of bat origin based on the most closely related coronavirus isolates [3-5], The virus was subsequently named SARS-CoV-2. SARS-CoV-2 was found to bind the same cell surface receptor used by SARS-CoV, angiotensin converting enzyme 2 (ACE2), via the receptor binding domain of the viral surface spike protein [6],

Previous studies have shown that antibodies derived from the memory B cells of recovered human patients can be used to protect against coronavirus diseases such as SARS and MERS in animal models [7, 8], In particular, antibodies that block binding of the viral spike protein to ACE2 have been shown to be highly effective at protecting against infection [9, 10], While such antibodies have not been used in the clinic, antibody therapeutics derived from recovered patients have been used to treat disease caused by infection with the highly lethal Ebola virus, with improved treatment outcomes relative to therapy with small molecule antiviral agents [11], Antibodies derived from recovered COVID19 patients might be useful for the treatment and prevention of disease caused by SARS-CoV-2 infection.

Summary

In a first aspect, the present disclosure provides an antigen-binding molecule, optionally isolated, which binds to SARS-CoV-2 spike protein.

In some embodiments, the antigen-binding molecule binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein.

In some embodiments, the antigen-binding molecule inhibits interaction between SARS-CoV-2 spike protein and ACE2. In some embodiments, the antigen-binding molecule inhibits infection of ACE2-expressing cells by SARS-CoV-2.

In some embodiments, the antigen-binding molecule comprises:

(a)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:56; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:10 LC-CDR2 having the amino acid sequence of SEQ ID NO:11 LC-CDR3 having the amino acid sequence of SEQ ID NO:12; or

(b)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:18; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

(c)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:4; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

(d)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NQ:20 HC-CDR2 having the amino acid sequence of SEQ ID NO:21 HC-CDR3 having the amino acid sequence of SEQ ID NO:22; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:27 LC-CDR2 having the amino acid sequence of SEQ ID NO:28 LC-CDR3 having the amino acid sequence of SEQ ID NO:29; or (e)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35 HC-CDR2 having the amino acid sequence of SEQ ID NO:58 HC-CDR3 having the amino acid sequence of SEQ ID NO:37; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:43 LC-CDR2 having the amino acid sequence of SEQ ID NO:44 LC-CDR3 having the amino acid sequence of SEQ ID NO:45; or

(f)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35 HC-CDR2 having the amino acid sequence of SEQ ID NO:36 HC-CDR3 having the amino acid sequence of SEQ ID NO:37; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

(g)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35 HC-CDR2 having the amino acid sequence of SEQ ID NO:51 HC-CDR3 having the amino acid sequence of SEQ ID NO:37; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

(h)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35 HC-CDR2 having the amino acid sequence of SEQ ID NO:54 HC-CDR3 having the amino acid sequence of SEQ ID NO:37; and

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:43 LC-CDR2 having the amino acid sequence of SEQ ID NO:44 LC-CDR3 having the amino acid sequence of SEQ ID NO:45.

In some embodiments, the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:55, 17, 1 , 19, 57, 34, 50 or 53; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9, 26 or 42.

In some embodiments, the antigen-binding molecule comprises:

(i) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:55; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9; or

(ii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:17; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9; or

(iii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1 ; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9; or

(iv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:57; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42; or

(v) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:19; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:26; or

(vi) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:34; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42; or

(vii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:50; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42; or (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:53; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42.

In some embodiments, the antigen-binding molecule comprises:

(i) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:62, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:61 ; or

(ii) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NQ:60, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:61 ; or

(iii) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:63, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:64; or

(iv) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:65, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66; or

(v) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:67, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66; or

(vi) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66.

The present disclosure also provides an antigen-binding molecule produced by the cell line MCB- 115-05, deposited 5 November 2020 as ATCC patent deposit number PT A-126858.

The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to the present disclosure.

The present disclosure also provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the present disclosure. The present disclosure also provides a cell comprising an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the present disclosure.

The present disclosure also provides a cell of the cell line designated MCB-115-05, deposited 5 November 2020 as ATCC patent deposit number PT A-126858.

The present disclosure also provides a method for producing an antigen-binding molecule which binds to SARS-CoV-2 spike protein, comprising culturing a cell according to the present disclosure under conditions suitable for expression of an antigen-binding molecule by the cell.

The present disclosure also provides a composition comprising an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The present disclosure also provides a composition comprising an antigen-binding molecule according to the present disclosure, wherein the composition comprises:

(i) 2 mM to 200 mM histidine, 2% to 20% (w/v) sucrose; 0.001 % to 0.1 % (w/v) polysorbate- 80, and has a pH 4.0 to 7.0; or

(ii) 2 mM to 200 mM histidine, 1 mM to 100 mM arginine, 2% to 20% (w/v) sucrose; 0.001 % to 0.1 % (w/v) polysorbate-80, and has a pH 4.0 to 7.0.

(iii) 1 mM to 100 mM acetate, 2% to 20% (w/v) sucrose; 0.001 % to 0.1 % (w/v) polysorbate- 80, and has a pH 4.0 to 7.0; or

(iv) 2 mM to 200 mM histidine, 1 mM to 50 mM methionine, 2% to 20% (w/v) sucrose; 0.001 % to 0.1 % (w/v) polysorbate-80, and has a pH 4.0 to 7.0.

In some embodiments, the composition comprises:

(i) 20 mM histidine, 8% (w/v) sucrose; 0.02% (w/v) polysorbate-80, and has a pH 6.0; or

(ii) 20 mM histidine, 10 mM arginine, 8% (w/v) sucrose; 0.02% (w/v) polysorbate-80, and has a pH 5.2; or

(iii) 10 mM acetate, 9% (w/v) sucrose; 0.01 % (w/v) polysorbate-80, and has a pH 5.2; or

(iv) 20 mM histidine, 8% (w/v) sucrose; 0.02% (w/v) polysorbate-80, and has a pH 5.2; or

(v) 20 mM histidine, 5 mM methionine, 8% (w/v) sucrose; 0.02% (w/v) polysorbate-80, and has a pH 5.2.

The present disclosure also provides an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in a method of medical treatment or prophylaxis. The present disclosure also provides an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in a method of treatment or prevention of a disease caused by infection with SARS-CoV-2.

The present disclosure also provides the use of an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, in the manufacture of a medicament for use in a method of treatment or prevention of a disease caused by infection with SARS-CoV-2.

The present disclosure also provides a method of treating or preventing a disease caused by infection with SARS-CoV-2, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure.

The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure to inhibit infection of ACE2-expressing cells by SARS-CoV-2.

The present disclosure also provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the present disclosure bound to SARS-CoV-2 spike protein.

The present disclosure also provides a method for detecting SARS-CoV-2 in a sample, comprising contacting a sample containing, or suspected to contain, SARS-CoV-2 with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex of the antigen-binding molecule with SARS-CoV-2 spike protein.

The present disclosure also provides a method for diagnosing a disease caused by infection with SARS-CoV-2, comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to the present disclosure and detecting the formation of a complex of the antigen-binding molecule with SARS-CoV-2 spike protein.

The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure in a method for detecting, localizing or imaging SARS-CoV-2, or cells infected with SARS-CoV-2.

The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure as an in vitro or in vivo diagnostic or prognostic agent. Description

The present disclosure provides antigen-binding molecules capable of binding to SARS-CoV-2 spike protein, in particular neutralising antibodies capable of inhibiting interaction between SARS-CoV-2 spike protein and ACE2, thus behaving as antagonists of infection of ACE2-expressing cells by SARS-CoV-2.Antigen-binding molecules described herein are provided with a combination of advantageous properties over known SARS-CoV-2 spike protein-binding antibodies.

SARS-CoV-2 spike protein

The present disclosure concerns severe acute respiratory syndrome-related coronavirus (SARSr- CoV). The virology of SARSr-CoV and epidemiology of disease associated with SARSr-CoV infection is reviewed, for example, in Cheng et al., Clin Microbiol Rev (2007) 20(4): 660-694 and de Wit et al., Nat Rev Microbiol (2016) 14: 523-534, both of which are hereby incorporated by reference in their entirety.

SARSr-CoV is a species of coronavirus of the genus Betacoronavirus and subgenus Sarbecoronavirus that infects humans, bats and certain other mammals. It is an enveloped positivesense single-stranded RNA virus.

Two strains of SARSr-CoV have caused serious outbreaks of severe respiratory diseases in humans: SARS-CoV, which caused an outbreak of severe acute respiratory syndrome (SARS) between 2002 and 2003, and SARS-CoV-2, which has caused the coronavirus disease 2019 (COVID-19) pandemic. There are hundreds of strains of SARSr-CoV known only to infect nonhuman species; bats are a major reservoir of many strains of SARS-related coronaviruses.

As used herein, “SARS-CoV-2” refers to the SARSr-CoV having the nucleotide sequence of GenBank: MN996527.1 (“Severe acute respiratory syndrome coronavirus 2 isolate WIV02, complete genome”), reported in Zhou et al., Nature (2020) 579: 270-273, and encompasses variants thereof having a nucleotide sequence with at least 85% sequence identity (e.g. one of at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater sequence identity) to the nucleotide sequence of GenBank: MN996527.1 .

Variants of SARS-CoV-2 of particular interest include: (i) the variant designated VUI-202012/01 , which belongs to the B.1 .1 .7 lineage, having the canonical nucleotide sequence of GISAID accession EPI_ISL_601443; (ii) the variant designated 501Y.V2/B.1 .351 , having the canonical nucleotide sequence of GISAID accession EPI_ISL_768642; (iii) the variant known as B.1.1.248/P.1 , having the canonical nucleotide sequence of GISAID accession EPI_ISL_792680; (iv) the variant known as B.1.617.1 , having the canonical nucleotide sequence of GISAID accession EPI_ISL_2621960; and (v) the variant known as B.1.617.2, having the canonical nucleotide sequence of GISAID accession EPI_ISL_1663476. The SARS-CoV-2 genome encodes four major structural proteins: the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.

The spike protein of SARS-CoV-2 has the amino acid sequence shown in SEQ ID NO:102. SARS- CoV-2 spike protein comprises S1 (SEQ ID NO:107) and S2 (SEQ ID NQ:110) subunits. The S1 subunit comprises a minimal receptor-binding domain (RBD; SEQ ID NQ:108) through which the SARSr-CoV binds to ACE2 expressed by host cells. The RBD in turn comprises the receptor binding motif (RBM; SEQ ID NQ:109), which is the region of the RBD that contacts ACE2.

In this specification “SARS-CoV-2 spike protein” refers to a polypeptide having the amino acid sequence shown in SEQ ID NQ:102, or polypeptide having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to SEQ ID NQ:102. Such polypeptides may include e.g. isoforms, fragments, variants of the spike protein encoded by SARS-CoV-2, and homologues from other SARSr-CoV (e.g. SARS-CoV).

Fragments of SARS-CoV-2 spike protein may have a minimum length of one of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, 1 ,100 or 1 ,200 amino acids, and may have a maximum length of one of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, 1 ,100 or 1 ,200 amino acids.

In this specification “the RBD of SARS-CoV-2 spike protein” refers to a polypeptide having the amino acid sequence shown in SEQ ID NQ:108, or polypeptide having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to SEQ ID NQ:108. Such polypeptides may include e.g. isoforms, fragments, variants of the RBD of the spike protein encoded by SARS-CoV-2, and the corresponding region of spike protein homologues from other SARSr-CoV.

Fragments of the RBD of SARS-CoV-2 spike protein may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200 amino acids, and may have a maximum length of one of 20, 10, 20, 30, 40, 50, 100, 150, 200 amino acids.

Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference protein, as determined by analysis by a suitable assay for the functional property/activity. For example, an isoform, fragment, variant or homologue of the spike protein of SARS-CoV-2 may display association with ACE2.

In some embodiments, the SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:102. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:104. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:107. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:108. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:109. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:120. In some embodiments, a fragment of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:121.

In some embodiments, a fragment of the RBD of SARS-CoV-2 spike protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:109.

Variants of SARS-CoV-2 spike protein have been reported. More frequently-detected mutations include D614G, N439K and S477N. Medium-frequency mutations include V483A, D839Y and T478I. Low frequency mutations include L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q and R403K. Very low frequency mutations include Q414E, I434K, S438F, K458N, D467V, I468F and V503F.

In some embodiments, a variant of a SARS-CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, V483A, D839Y, T478I, L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q, R403K, Q414E, I434K, S438F, K458N, D467V, I468F and V503F. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, V483A, D839Y, T478I, L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q and R403K. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, V483A, D839Y and T478I. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 , 2 or 3) of the following mutations: D614G, N439K and S477N.

In some embodiments, a variant of a SARS-CoV-2 spike protein according to the present disclosure may belong to, or be derived from, the SARS-CoV-2 B.1 .617 lineage e.g. B.1 .617.1 and/or B.1 .617.2 (including B.1.617.2.1 and B.1.617.2.2). In some embodiments, a variant of a SARS-CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: G142D, E154K, L452R, E484Q, D614G, P681 R, and Q1071 H, optionally in combination with any other mutations described herein. In some embodiments, a variant of a SARS- CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: T19R, G142D, EFR156-158G, L452R, T478K, D614G, P681 R, and D950N, optionally in combination with any other mutations described herein. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 or 2) of the following mutations: P681 R and L452R.

In some embodiments, a variant of the RBD of SARS-CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: N439K, S477N, V483A, T478I, V367F, G476S, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q, R403K, Q414E, I434K, S438F, K458N, D467V, I468F, V503F, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L and G496S. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: N439K, S477N, V483A, T478I, V367F, G476S, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q, R403K, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L and G496S. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 , 2, 3 or 4) of the following mutations: N439K, S477N, V483A, and T478I. In some embodiments, a variant of a SARS-CoV-2 spike protein comprises one or more (e.g. 1 or 2) of the following mutations: N439K and S477N.

In some embodiments, a variant of the RBD of SARS-CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 or 2) of the following mutations: L452R and E484Q, optionally in combination with one or more (e.g. 1 , 2, 3, 4, 5, etc.) mutations described herein. In some embodiments, a variant of the RBD of SARS-CoV-2 spike protein according to the present disclosure comprises one or more (e.g. 1 or 2) of the following mutations: L452R and T478K, optionally in combination with one or more (e.g. 1 , 2, 3, 4, 5, etc.) mutations described herein. Angiotensin-converting enzyme 2 (ACE2) is a single-pass type I transmembrane carboxypeptidase which attaches to the cell membrane of cells of the outer surface tissues of lungs, arteries, heart, kidney, and intestines. The structure and function of ACE2 is described e.g. in Hamming et al., J Pathol (2004) 203(2): 631-637, which is hereby incorporated by reference in its entirety. ACE2 has been identified to be the entry point into cells for SARS-CoV-2, via interaction with the spike protein; the SARS-CoV-2 spike protein binds to the extracellular domain of ACE2 (Zhou et al., Nature (2020) 579: 270-273; Hoffmann et al., Cell (2020) 181 : 271-280).

In this specification “ACE2” refers to ACE2 from any species and includes ACE2 isoforms, fragments, variants or homologues from any species. In some embodiments, the ACE2 is ACE2 from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the ACE2 is ACE2 from a human, bat, pangolin, civet or pig. Isoforms, fragments, variants or homologues of ACE2 may optionally be characterised as having at least 70% sequence identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature ACE2 isoform from a given species, e.g. human.

Human ACE2 isoform 1 is shown in SEQ ID NO:112, and human ACE2 isoform 2 is shown in SEQ ID NO:119. The extracellular domain of human ACE2 is shown in SEQ ID NO:114.

Fragments of ACE2 may have a minimum length of one of 25, 50, 100, 200, 300, 400, 500, 600, 700 or 800 amino acids, and may have a maximum length of one of 50, 100, 200, 300, 400, 500, 600, 700 or 800 amino acids. Fragments of ACE2 may e.g. display association with SARS-CoV-2 spike protein.

In some embodiments, the ACE2 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:112 or 119.

In some embodiments, a fragment of ACE2 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:114.

molecules

The present disclosure provides antigen-binding molecules capable of binding to SARS-CoV-2 spike protein. Antigen-binding molecules according to the present disclosure may be provided in purified or isolated form, i.e. from other naturally-occurring biological material. An “antigen-binding molecule” refers to a molecule which is capable of binding to a target antigen, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s).

The antigen-binding molecule of the present disclosure comprises a moiety or moieties capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of a antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

As used herein, a “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

The antigen-binding molecules of the present disclosure generally comprise an antigen-binding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen. The antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv region.

An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigen-binding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain. The polypeptides may associate covalently or non-covalently. In some embodiments the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).

An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides. The antigen-binding molecules of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to SARS-CoV-2 spike protein. Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used/provided. An “antigen-binding region” is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.

Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen.

The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC- CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C term.

There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671- D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

In some embodiments, the antigen-binding molecule comprises the CDRs of an antibody capable of binding to SARS-CoV-2 spike protein described herein, or comprises CDRs which are derived from an antibody capable of binding to SARS-CoV-2 spike protein described herein. In some embodiments, the antigen-binding molecule comprises the FRs of an antibody capable of binding to SARS-CoV-2 spike protein described herein, or comprises FRs which are derived from an antibody capable of binding to SARS-CoV-2 spike protein described herein. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antibody capable of binding to SARS-CoV-2 spike protein described herein, or comprises CDRs and FRs which are derived from an antibody capable of binding to SARS-CoV-2 spike protein described herein. That is, in some embodiments the antigen-binding molecule comprises the VH region and the VL region of an antibody capable of binding to SARS-CoV-2 spike protein described herein, or comprises VH and VL regions which are derived from an antibody capable of binding to SARS-CoV-2 spike protein described herein. In some embodiments the antigen-binding molecule comprises the CDRs, FRs and/or the VH and/or VL regions of an antibody capable of binding to SARS-CoV-2 spike protein selected from: SC31 WT, SC31 GS, SC31 GSeng, SC1 , SC1 GS, SC11 , SC1 1 GS, SC11 GSeng1 and SC11 GSeng2.

In some embodiments the antigen-binding molecule comprises a VH region according to one of (1) to (8) below:

(1) [SC31WT, SC31 GS, SC31 GSeng CON] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2

HC-CDR2 having the amino acid sequence of SEQ ID NO:3

HC-CDR3 having the amino acid sequence of SEQ ID NO:56, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid.

(2) [SC1 1 , SC11GS, SC11 GSeng1 , SC1 1 GSeng2 CON] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35

HC-CDR2 having the amino acid sequence of SEQ ID NO:58

HC-CDR3 having the amino acid sequence of SEQ ID NO:37, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid.

(3) [SC31WT, SC31 GS] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2

HC-CDR2 having the amino acid sequence of SEQ ID NO:3

HC-CDR3 having the amino acid sequence of SEQ ID NO:4, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid.

(4) [SC31 GSeng] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:2

HC-CDR2 having the amino acid sequence of SEQ ID NO:3

HC-CDR3 having the amino acid sequence of SEQ ID NO:18, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid.

(5) [SC1 , SC1 GS] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NQ:20

HC-CDR2 having the amino acid sequence of SEQ ID NO:21

HC-CDR3 having the amino acid sequence of SEQ ID NO:22, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid.

(6) [SC1 1 , SC11GS] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35

HC-CDR2 having the amino acid sequence of SEQ ID NO:36

(7) [SC1 1 GSeng1 ] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35

HC-CDR2 having the amino acid sequence of SEQ ID NO:51

(8) [SC1 1 GSeng2] a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:35

HC-CDR2 having the amino acid sequence of SEQ ID NO:54

In some embodiments the antigen-binding molecule comprises a VH region according to one of (9) to (13) below:

(9) [SC31WT, SC31 GS, SC31 GSeng CON; SC31WT, SC31 GS, SC31GSeng] a VH region incorporating the following FRs:

HC-FR1 having the amino acid sequence of SEQ ID NO:5

HC-FR2 having the amino acid sequence of SEQ ID NO:6

HC-FR3 having the amino acid sequence of SEQ ID NO:7

HC-FR4 having the amino acid sequence of SEQ ID NO:8, or a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

(10) [SC11 , SC11 GS, SC1 1 GSengl , SC11 GSeng2 CON] a VH region incorporating the following FRs:

HC-FR1 having the amino acid sequence of SEQ ID NO:38

HC-FR2 having the amino acid sequence of SEQ ID NO:39

HC-FR3 having the amino acid sequence of SEQ ID NO:59 HC-FR4 having the amino acid sequence of SEQ ID NO:41 , or a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

(11) [SC1 , SC1 GS] a VH region incorporating the following FRs:

HC-FR1 having the amino acid sequence of SEQ ID NO:23

HC-FR2 having the amino acid sequence of SEQ ID NO:24

HC-FR3 having the amino acid sequence of SEQ ID NO:25

(12) [SC11 , SC11 GS] a VH region incorporating the following FRs:

HC-FR1 having the amino acid sequence of SEQ ID NO:38

HC-FR2 having the amino acid sequence of SEQ ID NO:39

HC-FR3 having the amino acid sequence of SEQ ID NQ:40

HC-FR4 having the amino acid sequence of SEQ ID NO:41 , or a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

(13) [SC11 GSeng1 , SC11 GSeng2] a VH region incorporating the following FRs:

HC-FR1 having the amino acid sequence of SEQ ID NO:38

HC-FR2 having the amino acid sequence of SEQ ID NO:39

HC-FR3 having the amino acid sequence of SEQ ID NO:52

In some embodiments the antigen-binding molecule comprises a VH region comprising the CDRs according to one of (1), (2), (3), (4), (5), (6), (7) or (8) above, and the FRs according to one of (9), (10), (11), (12) or (13) above.

In some embodiments the antigen-binding molecule comprises a VH region according to one of (14) to (21) below:

(14) a VH region comprising the CDRs according to (1) and the FRs according to (9).

(15) a VH region comprising the CDRs according to (3) and the FRs according to (9).

(16) a VH region comprising the CDRs according to (4 and the FRs according to (9). (17) a VH region comprising the CDRs according to (2) and the FRs according to (10).

(18) a VH region comprising the CDRs according to (5) and the FRs according to (11).

(19) a VH region comprising the CDRs according to (6) and the FRs according to (12).

(20) a VH region comprising the CDRs according to (7) and the FRs according to (13).

(21) a VH region comprising the CDRs according to (8) and the FRs according to (13).

In some embodiments the antigen-binding molecule comprises a VH region according to one of (22) to (29) below:

(22) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:55.

(23) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:57.

(24) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:1 .

(25) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:17.

(26) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:19.

(27) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:34.

(28) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:50. (29) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:53.

In some embodiments the antigen-binding molecule comprises a VL region according to one of (30) to (32) below:

(30) [SC31WT, SC31 GS, SC31GSeng] a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:10

LC-CDR2 having the amino acid sequence of SEQ ID NO:11

LC-CDR3 having the amino acid sequence of SEQ ID NO:12, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, or LC-CDR3 are substituted with another amino acid.

(31) [SC1 , SC1GS] a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:27

LC-CDR2 having the amino acid sequence of SEQ ID NO:28

LC-CDR3 having the amino acid sequence of SEQ ID NO:29, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, or LC-CDR3 are substituted with another amino acid.

(32) [SC11 , SC11 GS, SC11 GSengl , SC11 GSeng2] a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:43

LC-CDR2 having the amino acid sequence of SEQ ID NO:44

LC-CDR3 having the amino acid sequence of SEQ ID NO:45, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, or LC-CDR3 are substituted with another amino acid.

In some embodiments the antigen-binding molecule comprises a VL region according to one of (33) to (35) below:

(33) [SC31WT, SC31 GS, SC31GSeng] a VL region incorporating the following FRs:

LC-FR1 having the amino acid sequence of SEQ ID NO:13

LC-FR2 having the amino acid sequence of SEQ ID NO:14

LC-FR3 having the amino acid sequence of SEQ ID NO:15

LC-FR4 having the amino acid sequence of SEQ ID NO:16, or a variant thereof in which one or two or three amino acids in one or more of LC-FR1 , LC-FR2, LC- FR3, or LC-FR4 are substituted with another amino acid.

(34) [SC1 , SC1GS] a VL region incorporating the following FRs:

LC-FR1 having the amino acid sequence of SEQ ID NQ:30

LC-FR2 having the amino acid sequence of SEQ ID NO:31 LC-FR3 having the amino acid sequence of SEQ ID NO:32

LC-FR4 having the amino acid sequence of SEQ ID NO:33, or a variant thereof in which one or two or three amino acids in one or more of LC-FR1 , LC-FR2, LC- FR3, or LC-FR4 are substituted with another amino acid.

(35) [SC11 , SC11 GS, SC11 GSengl , SC11 GSeng2] a VL region incorporating the following FRs:

LC-FR1 having the amino acid sequence of SEQ ID NO:46

LC-FR2 having the amino acid sequence of SEQ ID NO:47

LC-FR3 having the amino acid sequence of SEQ ID NO:48

LC-FR4 having the amino acid sequence of SEQ ID NO:49, or a variant thereof in which one or two or three amino acids in one or more of LC-FR1 , LC-FR2, LC- FR3, or LC-FR4 are substituted with another amino acid.

In some embodiments the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (30), (31) or (32) above, and the FRs according to one of (33), (34) or (35) above.

In some embodiments the antigen-binding molecule comprises a VL region according to one of (36) to (38) below:

(36) a VL region comprising the CDRs according to (30) and the FRs according to (33).

(37) a VL region comprising the CDRs according to (31) and the FRs according to (34).

(38) a VL region comprising the CDRs according to (32) and the FRs according to (35).

In some embodiments the antigen-binding molecule comprises a VL region according to one of (39) to (41) below:

(39) a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:9.

(40) a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:26.

(41) a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments the antigen-binding molecule comprises a VH region according to any one of (1) to (29) above, and a VL region according to any one of (30) to (41) above.

In embodiments in accordance with the present disclosure in which one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example according to the following Table. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same line in the rightmost column are substituted:

In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.

The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present disclosure comprises, or consists of, an Fv region which binds to SARS-CoV-2 spike protein. In some embodiments the VH and VL regions of the Fv may be provided as single polypeptide joined by a linker region, i.e. a single chain Fv (scFv).

The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA). In some embodiments the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide). In some embodiments the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments the Fab region is a CrossFab region. In some embodiments the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

In some embodiments, the antigen-binding molecule of the present disclosure comprises, or consists of, a Fab region which binds to SARS-CoV-2 spike protein. In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to SARS-CoV-2 spike protein. As used herein, “whole antibody” refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.

Immunoglobulins of type G (i.e. IgG) are ~150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chain comprise a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. Ig A1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

In some embodiments, the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to SARS-CoV- 2 spike protein.

In some embodiments the antigen-binding molecule of the present disclosure comprises one or more regions (e.g. CH1 , CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. h Ig A1 , hlgA2), hlgD, h IgE or hlgM. In some the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human IgG 1 allotype (e.g. G1 ml , G1 m2, G1 m3 or G1 ml 7).

In some embodiments the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:69 or 74.

In some embodiments the antigen-binding molecule comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NOTO or 75. In some embodiments the antigen-binding molecule comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:71. In some embodiments the antigen-binding molecule comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:72. In some embodiments the antigen-binding molecule comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:73 or 76.

In some embodiments the antigen-binding molecules of the present disclosure comprise an Fc region. An Fc region is composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.

Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated virus inhibition (ADCVI), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation. Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc- mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region. Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations. To improve the yield of the desired combinations of polypeptides in antigen-binding molecules in recombinant production, it is advantageous to introduce in the Fc regions modification(s) promoting association of the desired combination of heavy chain polypeptides. Modifications may promote e.g. hydrophobic and/or electrostatic interaction between CH2 and/or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety. In some embodiments the antigen-binding molecule of the present disclosure comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of formats shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394.

In some embodiments the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC;

CK). In some embodiments the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.

In some embodiments the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:77, 78, 79, 80, 81 or 82.

Multispecific antigen-binding molecules are also contemplated. By “multispecific” it is meant that the antigen-binding molecule displays specific binding to more than one target. In some embodiments the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).

In some embodiments the antigen-binding molecule binds to SARS-CoV-2 spike protein and another target (e.g. an antigen other than SARS-CoV-2 spike protein), and so is at least bispecific. The term “bispecific” means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.

It will be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific. For example, an antigen-binding molecule which is capable of binding to SARS-CoV-2 spike protein and an antigen other than SARS- CoV-2 spike protein may comprise: (i) an antigen-binding molecule which is capable of binding to SARS-CoV-2 spike protein, and (ii) an antigen-binding molecule which is capable of binding to an antigen other than SARS-CoV-2 spike protein.

It will also be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding polypeptides or antigenbinding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific. In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g. a multispecific antigen-binding molecule) may be referred to e.g. as an “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.

In some embodiments the antigen other than SARS-CoV-2 spike protein in a multispecific antigenbinding molecule is an immune cell surface molecule. In some embodiments the antigen is a receptor molecule, e.g. a cell surface receptor. In some embodiments the antigen is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine. In some embodiments the antigen is a growth factor or a hormone. An immune cell surface molecule may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell. In some embodiments, the part of the immune cell surface molecule which is bound by the antigen-binding molecule of the present disclosure is on the external surface of the immune cell (i.e. is extracellular). The immune cell surface molecule may be expressed at the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell).

Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1/CL fusion proteins, e.g. scFv2-CH1/CL, VHH2-CH1/CL; “variable domain only” bispecific antigen-binding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb/VHH, tertravalent dAb.VHH; Non-lg fusion proteins, e.g. scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g. scFv-Fc(kih), scFv-Fc(CH3 charge pairs), scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEEDbody), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv- Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-lg, scFv2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE/IgM CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab- dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-lgG(kih), Fab-scFab- IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1/CL charge pairs, hinge/CH3 charge pairs, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab- IgG, FcFc*; appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab- Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv- Fc-SEEDbody, TriFab; appended IgGs - HC fusions, e.g. IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG- CrossFab, IgG-orthogonal Fab, IgG-(CaCP) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab) Fab-lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3); appended IgGs - LC fusions, e.g. IgG- scFv(LC), scFv(LC)-lgG, dAb-IgG; appended IgGs - HC and LC fusions, e.g. DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody; Fc fusions, e.g. Fab-scFv-Fc, scFv4-lg; F(ab’)2 fusions, e.g. F(ab’)2- SCFV2; CH1/CL fusion proteins e.g. scFv2-CH1-hinge/CL; modified IgGs, e.g. DAF (two-in one-IgG), DutaMab, Mab²; and non-lg fusions, e.g. DNL-Fab4-lgG.

The skilled person is able to design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemically crosslinking of antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 - 2.13.16, which is hereby incorporated by reference in its entirety. For example, /V-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide- linked bispecific F(ab)2 heterodimers.

Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antigenbinding molecules. Current Protocols in Immunology. 14:l V:2.13:2.13.1 - 2.13.16. Other methods include recombinant expression for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigenbinding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigen-binding molecules, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.

Particular exemplary polypeptides and antiqen-bindinq molecules

The present disclosure also provides polypeptide constituents of antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.

The antigen-binding molecule of the present disclosure may be, or may comprise, a complex of polypeptides.

In the present specification where a polypeptide comprises more than one domain or region, it will be appreciated that the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains/regions.

In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VL as described herein.

In some embodiments, the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).

In some embodiments the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the polypeptide comprises a CH1 region as described herein. In some embodiments the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments the polypeptide comprises a CH2 region as described herein. In some embodiments the polypeptide comprises a CH3 region as described herein. In some embodiments the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the polypeptide comprises a CL region as described herein.

In some embodiments, the polypeptide according to the present disclosure comprises a structure from N- to C-terminus according to one of the following:

(i) VH

(ii) VL

(iii) VH-CH1

(iv) VL-CL

(v) VH-CH1-CH2-CH3

Also provided by the present disclosure are antigen-binding molecules composed of the polypeptides of the present disclosure. In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:

(A) VH + VL

(B) VH-CH1 + VL-CL

(C) VH-CH1-CH2-CH3 + VL-CL

In some embodiments the antigen-binding molecule comprises more than one of a polypeptide of the combinations shown in (A) to (C) above. By way of example, with reference to (C) above, in some embodiments the antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.

It will be appreciated in the context of (i) to (v) and (A) to (C) above that “VH” may refer to a VH region according to any one of (1) to (29) described herein, and “VL” may refer to a VL region according to any one of (30) to (341) described herein.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 , 17, 19, 34, 50 or 53.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:9, 26 or 42.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:60, 62, 63, 65, 67 or 68.

In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:61 , 64 or 66.

In some embodiments, the antigen-binding molecule of the present disclosure comprises:

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 , and

(ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:9.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:17, and

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:19, and (ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:26.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:34, and

(ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:42.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:50, and

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:53, and

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:60, and

(ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:61.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:62, and (ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:61.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:63, and

(ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:64.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:65, and

(ii) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:66.

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:67, and

(i) a polypeptide which comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:68, and

Linkers and additional sequences

In some embodiments the antigen-binding molecules and polypeptides of the present disclosure comprise one or more linker sequences between amino acid sequences. A linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule/polypeptide.

Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence comprises one or more copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide. For example, the antigen-binding molecule/polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide. In some embodiments the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.

The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.

The signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide. The signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigenbinding molecule/polypeptide secreted from the cell expressing the antigen-binding molecule/polypeptide. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

Labels and conjugates

In some embodiments the antigen-binding molecules of the present disclosure additionally comprise a detectable moiety.

In some embodiments the antigen-binding molecule comprises a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The antigen-binding molecule may be covalently or non-covalently labelled with the detectable moiety.

Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Iodine¹²³, Iodine¹²⁵, Iodine¹²⁶, Iodine¹³¹, Iodine¹³³, Bromine⁷⁷, Technetium^99m, Indium¹¹¹, lndium^113m, Gallium⁶⁷, Gallium⁶⁸, Ruthenium⁹⁵, Ruthenium⁹⁷, Ruthenium¹⁰³, Ruthenium¹⁰⁵, Mercury²⁰⁷, Mercury²⁰³, Rhenium^99m, Rhenium¹⁰¹, Rhenium¹⁰⁵, Scandium⁴⁷, Tellurium^121m, Tellurium^122m, Tellurium^125m, Thulium¹⁶⁵, Thuliuml¹⁶⁷, Thulium¹⁶⁸, Copper⁶⁷, Fluorine¹⁸, Yttrium⁹⁰, Palladium¹⁰⁰, Bismuth²¹⁷ and Antimony²¹¹. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers. Enzymatic labels include e.g. peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.

In some embodiments the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. Antibodydrug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14. In some embodiments, the chemical moiety may be a drug moiety (e.g. a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD. Functional properties of the antiqen-bindinq molecules

The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, the antigen-binding molecule described herein may possess one or more of the following properties: binds to SARS-CoV-2 spike protein; binds to the RBD of SARS-CoV-2 spike protein; binds to variants of SARS-CoV-2 spike protein; binds to the RBD of variants of SARS-CoV-2 spike protein; binds to SARS-CoV-2 spike protein from variant B.1.617.1 and/or B.1.617.2; binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein at low pH; inhibits interaction between SARS-CoV-2 spike protein and a ligand for SARS-CoV-2 spike protein (e.g. ACE2); inhibits infection of ACE2-expressing cells by SARS-CoV-2; reduces the level of SARS-CoV-2 in a subject infected with SARS-CoV-2 (e.g. in the lung); reduces the expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in a subject infected with SARS-CoV-2 (e.g. in the lung); reduces the number/proportion of cells comprising/infected with SARS-CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein; potentiates cell killing of cells comprising/infected with SARS-CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein; directs an Fc-mediated effector function (e.g. one or more of ADCC, ADCP, CDC, ADCVI, MAC complex formation or cell degranulation) against cells comprising/infected with SARS- CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein; binds to CD16a; binds to CD32a; binds to CD32b; binds to CD64; does not increase infection of cells by SARS-CoV-2 (i.e. through antibody-dependent enhancement); reduces the severity of symptoms of a disease caused by SARS-CoV-2 infection in a subject infected with SARS-CoV-2; increases survival of a subject infected with SARS-CoV-2; displays dose-dependent therapeutic efficacy in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection.

The antigen-binding molecules and antigen-binding domains described herein preferably display specific binding to a SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein, including variants thereof. As used herein, “specific binding” refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen. An antigen-binding molecule/domain that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.

The ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigenbinding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.

In some embodiments, the extent of binding of the antigen-binding molecule to a non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA. Alternatively, binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1 x 10ⁿ, where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

The affinity of binding to a given target antigen for an antigen-binding molecule described herein may be determined e.g. by Bio-Layer Interferometry, e.g. as described in Lad et al., (2015) J Biomol Screen 20(4): 498-507.

In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with sub-micromolar affinity, i.e. KD < 1 x 10⁶ M. In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with an affinity in the nanomolar range, i.e. KD = 9.9 x 10⁷ to 1 x 10 ⁹ M. In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with sub- nanomolar affinity, i.e. KD < 1 x 10⁹ M. In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with an affinity in the picomolar range, i.e. KD = 9.9 X 10 ¹⁰ to 1 x 10 ¹² M. In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with sub-picomolar affinity, i.e. KD < 1 x 10 ¹² M.

In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with a KD of 5 pM or less, preferably one of <5 pM, <2 pM, <1 pM, <500 nM, <100 nM, <75 nM, <50 nM, <40 nM, <30 nM, <20 nM, <15 nM, <12.5 nM, <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM <3 nM, <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <75 pM, <50 pM, <45 pM, <40 pM, <35 pM, <30 pM, <25 pM, <20 pM, <15 pM or <10 pM. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with an affinity of KD = <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <75 pM, <50 pM, <45 pM, <40 pM, <35 pM, <30 pM, <25 pM, <20 pM, <15 pM or <10 pM.

In some embodiments, the antigen-binding molecule described herein binds to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein with an affinity of binding (e.g. as determined by ELISA) of EC50 = <20 pg/ml, <10 pg/ml, <9 pg/ml, <8 pg/ml, <7 pg/ml, <6 pg/ml, <5 pg/ml, <4 pg/ml, <3 pg/ml, <2 pg/ml, <1.5 pg/ml, <1 pg/ml, <0.9 pg/ml, <0.8 pg/ml, <0.7 pg/ml, <0.6 pg/ml, <0.5 pg/ml, <0.4 pg/ml, <0.3pg/ml, <0.4 pg/ml, or <0.1 pg/ml.

In some embodiments, the antigen-binding molecule described herein retains binding to SARS-CoV- 2 spike protein at low pH. In some embodiments, the antigen-binding molecule described herein displays binding to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein at pH <7.0. In some embodiments, the antigen-binding molecule displays binding to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein at pH <7.0 and >3.0, e.g. pH <7.0 and >4.5. In some embodiments, the antigen-binding molecule displays binding to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein at pH 3.5, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9.

In some embodiments, the antigen-binding molecule described herein retains binding to SARS-CoV- 2 spike protein and/or the RBD of SARS-CoV-2 spike protein with similar affinity at low pH as it does at physiological pH (e.g. pH ~7.4). In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein at pH <7.0 and >3.0 (e.g. pH <7.0 and >4.5, e.g. pH 3.5, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9) with a KD value/EC50 value which is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the KD value/EC50 value for binding to SARS-CoV-2 spike protein and/or the RBD of SARS-CoV-2 spike protein at physiological pH (e.g. pH ~7.4).

In some embodiments, the antigen-binding molecule described herein binds to a variant of a SARS- CoV-2 spike protein. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, G142D, E154K, E484Q, P681 R, Q1071 H, T19R, EFR156-158G, D950N, V483A, D839Y, T478I, L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q, R403K, Q414E, I434K, S438F, K458N, D467V, I468F or V503F. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, G142D, E154K, E484Q, P681 R, Q1071 H, T19R, EFR156-158G, D950N, V483A, D839Y, T478I, L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, A475V, S494P, P479S, N501Y, V483F, P463S, S477I, E484K, F490S, L455F, F490L, E484Q or R403K. In some embodiments, the antigenbinding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, G142D, E154K, E484Q, P681 R, Q1071 H, T19R, EFR156-158G, D950N, V483A, D839Y or T478I. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5 etc) of the following mutations: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, G142D, E154K, E484Q, P681 R, Q1071 H, T19R, EFR156-158G, or D950N. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, or 3) of the following mutations: D614G, N439K, or S477N). In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 or 2) of the following mutations: P681 R and L452R.

In some embodiments, the antigen-binding molecule binds to the spike protein (and/or the RBD thereof) of the SARS-CoV-2 variant known as B.1.617.1. In some embodiments, the antigen-binding molecule binds to the spike protein (and/or the RBD thereof) of the SARS-CoV-2 variant known as B.1.617.2.

In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: G142D, E154K, L452R, E484Q, D614G, P681 R, and Q1071 H, optionally in combination with any other mutations described herein.

In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations: T19R, G142D, EFR156-158G, L452R, T478K, D614G, P681 R, and D950N, optionally in combination with any other mutations described herein.

In some embodiments, the antigen-binding molecule described herein binds to a variant of a SARS- CoV-2 spike protein - e.g. a variant of a SARS-CoV-2 spike protein described in the preceding paragraphs - with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NO:102. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NO:102: D614G, N439K, S477N, K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, G496S, G142D, E154K, E484Q, P681 R, Q1071 H, T19R, EFR156-158G, D950N, V483A, D839Y, T478I, L8V, H49Y, Q239K, S254F, V367F, G476S, S943R, S943T, R408I, G446V, S494P, P479S, V483F, P463S, S477I, E484K, F490S, F490L, E484Q, R403K, Q414E, K458N, I468F or V503F.

In some embodiments, the antigen-binding molecule binds to a spike protein of a SARS-CoV-2 variant of the lineage B.1 .617, e.g. B.1 .617.1 and/or B.1 .617.2, with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NO:102. In some embodiments, the antigen-binding molecule binds to a spike protein of a SARS-CoV-2 variant of the lineage B.1 .617, e.g. B.1 .617.1 , comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NO:102: G142D, E154K, L452R, E484Q, D614G, P681 R, or Q1071 H. In some embodiments, the antigen-binding molecule binds to a spike protein of a SARS-CoV-2 variant of the lineage B.1 .617, e.g. B.1 .617.2, comprising one or more (e.g. 1 , 2, 3, 4, 5, etc.) of the following mutations with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NQ:102: T19R, G142D, EFR156-158G, L452R, T478K, D614G, P681 R, or D950N. In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein comprising one or more (e.g. 1 or 2) of the following mutations with an affinity which is similar to the affinity to which it binds to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NQ:102: P681 R and L452R.

An antigen-binding molecule which binds to a variant of a SARS-CoV-2 spike protein with an affinity which is “similar” to the affinity to which it binds SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NQ:102 may bind to the variant with KD or EC50 value which is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the KD value/EC50 value for binding to SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NQ:102.

The antigen-binding molecules of the present disclosure may bind to a particular region of interest of the target antigen. The antigen-binding region of an antigen-binding molecule according to the present domain may bind to linear epitope of a target antigen, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). In some embodiments, the antigen-binding region molecule may bind to a conformational epitope of a target antigen, consisting of a discontinuous sequence of amino acids of the amino acid sequence. The region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based “protection” methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety.

In some embodiments the antigen-binding molecule is capable of binding to the region of SARS- CoV-2 spike protein shown in SEQ ID NO:104. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:104. In some embodiments the antigen-binding molecule is capable of binding to the region of SARS-CoV-2 spike protein shown in SEQ ID NQ:107. In some embodiments the antigenbinding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:107. In some embodiments the antigen-binding molecule is capable of binding to the region of SARS-CoV-2 spike protein shown in SEQ ID NQ:108. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:108. In some embodiments the antigen-binding molecule is capable of binding to the region of SARS-CoV-2 spike protein shown in SEQ ID NO:109. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:109.

In some embodiments the antigen-binding molecule is capable of binding to SARS-CoV-2 spike protein in the region comprising I434, S438, L455, D467, A475 and N501 (numbered according to SEQ ID NQ:102). In some embodiments the antigen-binding molecule is capable of binding to SARS-CoV-2 spike protein in the region comprising I434, S438, and D467. In some embodiments the antigen-binding molecule binds to SARS-CoV-2 spike protein via association through one or more of I434, S438, L455, D467, A475 and N501 . In some embodiments the antigen-binding molecule binds to SARS-CoV-2 spike protein via association through one or more of I434, S438, and D467.

In some embodiments the antigen-binding molecule is capable of binding to SARS-CoV-2 spike protein via interaction with the region of SARS-CoV-2 spike protein shown in SEQ ID NQ:120. In some embodiments the antigen-binding molecule is capable of binding to the region of SARS-CoV-2 spike protein shown in SEQ ID NQ:120. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:120.

In some embodiments the antigen-binding molecule is capable of binding to SARS-CoV-2 spike protein via interaction with the region of SARS-CoV-2 spike protein shown in SEQ ID NO:121 . In some embodiments the antigen-binding molecule is capable of binding to the region of SARS-CoV-2 spike protein shown in SEQ ID NO:121. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:121.

The ability of an antigen-binding molecule to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, surface plasmon resonance and biolayer interferometry.

In some embodiments the antigen-binding molecule is capable of binding the same region of SARS- CoV-2 spike protein, or an overlapping region of SARS-CoV-2 spike protein, to the region of SARS- CoV-2 spike protein which is bound by an antibody comprising a the VH and VL regions of one of SC31WT, SC31GS, SC31 GSeng, SC1 , SC1GS, SC11 , SC11GS, SC11GSeng1 and SC11GSeng2 described herein. In some embodiments the antigen-binding molecule is capable of binding the same region of SARS-CoV-2 spike protein, or an overlapping region of SARS-CoV-2 spike protein, to the region of SARS-CoV-2 spike protein which is bound by the antigen-binding molecule produced by the cell line designated MCB-115-05, deposited 5 November 2020 as ATCC patent deposit number PT A-126858.

Whether a test antigen-binding molecule binds to the same or an overlapping region of a given target as a reference antigen-binding molecule can be evaluated, for example, by analysis of (i) interaction between the test antigen-binding molecule and the target in the absence of the reference binding molecule, and (ii) interaction between the test antigen-binding molecule in the presence of the reference antigen-binding molecule, or following incubation of the target with the reference antigen-binding molecule. Determination of a reduced level of interaction between the test antigenbinding molecule and the target following analysis according to (ii) as compared to (i) might support an inference that the test and reference antigen-binding molecule bind to the same or an overlapping region of the target. Suitable assays for such analysis include e.g. competition ELISA assays and epitope binning assays.

In some embodiments, the antigen-binding molecule binds to SARS-CoV-2 spike protein in the region which is bound by an interaction partner for SARS-CoV-2 spike protein, e.g. ACE2. In some embodiments, the antigen-binding molecule inhibits interaction between an interaction partner for SARS-CoV-2 spike protein (e.g. ACE2) and SARS-CoV-2 spike protein. In some embodiments, the antigen-binding molecule is a competitive inhibitor of binding of an interaction partner for SARS- CoV-2 spike protein (e.g. ACE2) to SARS-CoV-2 spike protein. In some embodiments, the antigenbinding molecule binds to SARS-CoV-2 spike protein in the region bound by a polypeptide comprising or consisting of the sequence shown in SEQ ID NO:112, 114 or 119. Antigen-binding molecules which inhibit interaction between ACE2 and SARS-CoV-2 spike protein may be described as antagonists of such interaction, and may be referred to as neutralising antigenbinding molecules to SARS-CoV-2.

The ability of an antigen-binding molecule to inhibit interaction between two factors can be determined for example by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the antibody/fragment. An example of a suitable assay to determine whether a given antigen-binding molecule inhibits interaction between two interaction partners is a competition ELISA assay. An antigen-binding molecule which inhibits a given interaction (e.g. between SARS-CoV-2 spike protein and ACE2) is identified by the observation of a reduction/decrease in the level of interaction between the interaction partners in the presence of - or following incubation of one or both of the interaction partners with - the antigen-binding molecule, as compared to the level of interaction in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule). Suitable analysis can be performed in vitro, e.g. using recombinant interaction partners or using cells expressing the interaction partners. Cells expressing interaction partners may do so endogenously, or may do so from nucleic acid introduced into the cell. For the purposes of such assays, one or both of the interaction partners and/or the antigen-binding molecule may be labelled or used in conjunction with a detectable entity for the purposes of detecting and/or measuring the level of interaction.

The ability of an antigen-binding molecule to inhibit interaction between SARS-CoV-2 spike protein and ACE2 can be analysed as described in Example 2.3. The ability of an antigen-binding molecule to inhibit interaction between two binding partners can also be determined by analysis of the downstream functional consequences of such interaction, e.g. infection of ACE2-expressing cells by SARS-CoV-2.

In some embodiments, the antigen-binding molecule of the present disclosure inhibits interaction between SARS-CoV-2 spike protein and ACE2 to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of interaction between SARS-CoV-2 spike protein and ACE2 in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

In some embodiments the antigen-binding molecule inhibits infection of ACE2-expressing cells by SARS-CoV-2. Such antigen-binding molecules may be described as antagonising infection of ACE2- expressing cells, or may be referred to as neutralising antigen-binding molecules to SARS-CoV-2.

The ability of an antigen-binding molecule to inhibit infection of ACE2-expressing cells by SARS- CoV-2 can be analysed by detecting/quantifying infection of ACE2-expressing cells by SARS-CoV-2 in the presence of the antigen-binding molecule, and comparing the level of infection to the level observed in the absence of the antigen-binding molecule (and/or the level observed in presence of an appropriate control antigen-binding molecule). The ability of an antigen-binding molecule to inhibit infection of ACE2-expressing cells by SARS-CoV-2 can be analysed as described in Example 2.3.

In some embodiments, the antigen-binding molecule of the present disclosure inhibits infection of ACE2-expressing cells by SARS-CoV-2 to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of infection of ACE2-expressing cells by SARS-CoV-2 observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing the level of SARS-CoV-2 in a subject infected with SARS-CoV-2. In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing the viral load of SARS- CoV-2 in a subject infected with SARS-CoV-2. In some embodiments, the antigen-binding molecule is capable of reducing the level of SARS-CoV-2 in the lungs of a subject infected with SARS-CoV-2.

In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing the level of expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in a subject infected with SARS-CoV-2. In some embodiments, the antigen-binding molecule is capable of reducing the level of expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in the lungs of a subject infected with SARS-CoV-2.

In some embodiments, the antigen-binding molecule is able to inhibit infection of ACE2-expressing cells by SARS-CoV-2 independently of Fc- mediated function. In some embodiments, the antigenbinding molecule is able to inhibit infection of ACE2-expressing cells by SARS-CoV-2 by a mechanism not requiring binding of the antigen-binding molecule to an Fc receptor.

In some embodiments, the antigen-binding molecule is able to reduce the level of expression of a proinflammatory factor (e.g. IL-6) independently of Fc-mediated function. In some embodiments, the antigen-binding molecule is able to reduce the level of expression of a proinflammatory factor (e.g. IL-6) by a mechanism not requiring binding of the antigen-binding molecule to an Fc receptor.

Whether an antigen-binding molecule achieves a given functional effect by a mechanism requiring/involving Fc-mediated function can be evaluated e.g. by analysing the ability of the antigen-binding molecule provided in a format lacking a functional Fc region to achieve the given functional effect. For example, the ability to achieve the given functional effect can be investigated using an antigen-binding molecule comprising a ‘silent’ Fc region (e.g. comprising LALA substitutions), or using an antigen-binding molecule provided in a format lacking an Fc region (e.g. scFv, Fab etc.).

Herein, “expression” may refer to gene or protein expression. Gene and protein expression can be evaluated by means well known to the skilled person. Expression of a gene can be investigated e.g. by analysing the level of RNA transcribed from the gene by techniques such as RT-qPCR, northern blot, etc. Protein expression investigated e.g. using antibody-based methods including western blot, immunohisto/cytochemistry, flow cytometry, ELISA, ELISPOT, or by reporter-based methods.

The ability of antigen-binding molecule to reduce the level of/viral load of SARS-CoV-2/level of expression of a proinflammatory factor in a subject infected with SARS-CoV-2 or in an organ of a subject infected with SARS-CoV-2 may be evaluated e.g. in an appropriate in vivo model of SARS- CoV-2 infection, such as a model employing transgenic mice expressing human ACE2 described e.g. in Bao et al. J Infect Dis (2020) 222:551-555 and Bao et al., Nature (2020) 583:830-833. For example, analysis may be performed as described in Example 4.3.

Analysis may involve infecting subjects with SARS-CoV-2, administering an effective amount of the antigen-binding molecule, subsequently detecting/quantifying the level of SARS-CoV-2/expression of the proinflammatory factor in the subject or organ(s) of the subject (e.g. the lungs), and comparing the level detected to the level observed in the absence of treatment with the antigen-binding molecule (and/or the level observed following treatment with an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule is capable of reducing the level of/viral load of SARS-CoV-2/level of expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level/load observed in the absence of treatment with the antigen-binding molecule (or observed following treatment with an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule according to the present disclosure may potentiate (i.e. upregulate, enhance) cell killing of cells comprising/infected with SARS-CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein.

In some embodiments the antigen-binding molecule is capable of reducing the number/proportion of cells comprising/infected with SARS-CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein. In some embodiments, the antigen-binding molecule is capable of depleting/enhancing depletion of such cells. Antigen-binding molecules according to the present disclosure may comprise one or more moieties for potentiating a reduction in the number/proportion of cells comprising/infected with SARS-CoV-2, or cells comprising/expressing SARS-CoV-2 spike protein. For example, an antigen-binding molecule according to the present disclosure may e.g. comprise an Fc region and/or a drug moiety.

Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects. IgG Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc-mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation. Fc-mediated antiviral effects include antibody-dependent cell-mediated virus inhibition (ADCVI), which is described e.g. in Forth and Moog, Curr Opin HIV AIDS (2009) 4(5): 388- 393 (hereby incorporated by reference in its entirety).

In some embodiments, the antigen-binding molecule according to the present disclosure comprises an Fc region and is capable of potentiating/directing one or more of ADCC, ADCP, CDC or ADCVI against, and/or potentiating formation of a MAC on or cell degranulation of, a cell comprising or infected with SARS-CoV-2, or a cell comprising or expressing SARS-CoV-2 spike protein. In some embodiments, the antigen-binding molecule comprises an Fc region and is capable of potentiating/directing ADCC and/or CDC against a cell comprising or infected with SARS-CoV-2, or a cell comprising or expressing SARS-CoV-2 spike protein.

Fc-mediated functions such as ADCC and CDC activity can be analysed e.g. according to the methods described in Yamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporated by reference in its entirety), by ⁵¹Cr release assay as described e.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated by reference in its entirety), using the Pierce LDH Cytotoxicity Assay Kit, or as described in Example 7 herein. CDC can be analysed e.g. using a C1q binding assay, e.g. as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457- 466 (hereby incorporated by reference in its entirety).

In some embodiments the antigen-binding molecule binds to human CD16a, human CD32a, human CD32b, and/or human CD64. Antigen-binding molecules according to the present disclosure may comprise one or more moieties providing for binding to CD16a, CD32a, CD32b and/or CD64. For example, an antigen-binding molecule according to the present disclosure may e.g. comprise an Fc region capable of mediating binding of the antigen-binding molecule to CD16a, CD32a, CD32b and/or CD64.

The ability of a given antigen-binding molecule to bind to CD16a, CD32a, CD32b and/or CD64 can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay.

In some embodiments, the antigen-binding molecule of the present disclosure does not cause substantial antibody-dependent enhancement of infection of cells by SARS-CoV-2. Antibodydependent enhancement (ADE) is described e.g. in Arvin et al., Nature (2020) 584: 53-363 (hereby incorporated by reference in its entirety). In the case of viral disease, antibodies bound to a virus may engage Fc receptors expressed by cells through their Fc region, which may in turn facilitate infection of those cells, e.g. through enhancing viral attachment to/fusion with the cell membrane. ADE can result in a greater level of infection, and consequently more severe disease.

In some embodiments, the antigen-binding molecule does not increase infection of cells by SARS- CoV-2 (e.g. as compared to the level of infection in the absence of the antigen-binding molecule). In some embodiments, the antigen-binding molecule comprises an Fc region and does not increase infection of cells by SARS-CoV-2 (e.g. as compared to the level of infection in the absence of the antigen-binding molecule).

The ability of an antigen-binding molecule according to the present disclosure to cause ADE of infection of cells by SARS-CoV-2 can be analysed e.g. as described in Example 7 herein.

In some embodiments, the antigen-binding molecule is capable of increasing survival of a subject infected with SARS-CoV-2. The ability of antigen-binding molecule to increase survival of a subject infected with SARS-CoV-2 may be evaluated in a model employing transgenic mice expressing human ACE2 described above. For example, analysis may be performed as described in Example 4.3.

Analysis may involve infecting subjects with SARS-CoV-2, administering an effective amount of the antigen-binding molecule, monitoring survival of subjects, and comparing the survival of subjects administered with the antigen-binding molecule to the survival of untreated subjects (and/or the survival of subjects treated with an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule is capable of increasing survival of a subject infected with SARS-CoV-2 to greater than 1 times, e.g. one of >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the survival observed in the absence of treatment with the antigen-binding molecule (or observed following treatment with an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule described herein displays dose-dependent therapeutic efficacy in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection described e.g. in Bao et al. J Infect Dis (2020) 222:551-555, Bao et al., Nature (2020) 583:830-833 and Examples 4.3 and 7 herein.

In some embodiments, the antigen-binding molecule is able to reduce the level of SARS-CoV-2 in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection when administered at a concentration of >5 mg/kg bodyweight (e.g. > 5 mg/kg and < 20 mg/kg, e.g. one of 5 mg/kg, 10 mg/kg or 20 mg/kg bodyweight) within 48 hours of infection.

In some embodiments, the antigen-binding molecule is able to reduce the expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection when administered at a concentration of >5 mg/kg bodyweight (e.g. > 5 mg/kg and < 20 mg/kg, e.g. one of 5 mg/kg, 10 mg/kg or 20 mg/kg bodyweight) within 48 hours of infection.

In some embodiments, the antigen-binding molecule is able to reduce the severity of symptoms in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection when administered at a concentration of > 5 mg/kg bodyweight (e.g. > 5 mg/kg and < 20 mg/kg, e.g. one of 5 mg/kg, 10 mg/kg or 20 mg/kg bodyweight) within 48 hours of infection.

In some embodiments, the antigen-binding molecule is able to increase survival in the K18-hACE2 mouse model of disease caused by SARS-CoV-2 infection when administered at a concentration of > 5 mg/kg bodyweight (e.g. > 5 mg/kg and < 20 mg/kg, e.g. one of 5 mg/kg, 10 mg/kg or 20 mg/kg bodyweight) within 48 hours of infection.

In some embodiments treatment of a subject with SARS-CoV-2 infection with the antigen-binding molecule may be associated with one or more of the following (e.g. as compared to untreated subjects):

• Reduction in SARS-CoV-2 viral load;

• Reduction in SARS-CoV-2 viral load from Day 1 to Days 5, 8, 11 , and/or 15 (± 1 day) after treatment;

• Reduction in progression of SARS-CoV-2 viral infection as defined by COVID-19 related mechanical ventilation and/or supplemental oxygen, COVID-19 related new hospitalization (defined as >24 hours of acute care), COVID-19 related emergency room visit, and/or death by Day 29 after treatment; • Reduction in the proportion (percentage) of subjects who experience progression of viral infection as defined by COVID-19 related mechanical ventilation and/or supplemental oxygen, COVID-19 related new hospitalization (defined as >24 hours of acute care), COVID- 19 related emergency room visit, and/or death by Day 29 after treatment;

• Reduced time to symptom resolution in a subject infected with SARS-CoV-2;

• Increased proportion (percentage) of subjects who demonstrate symptom resolution;

• Reduced proportion (percentage) of subjects with detectable SARS-CoV-2, e.g. from Day 1 to Days 3, 5, 8, 11 , 15 and/or 29 after treatment;

• Increased survival of a subject infected with SARS-CoV-2;

• Reduced 15-day risk of mortality for a subject infected with SARS-CoV-2; and/or

• Reduced 28-day risk of mortality for a subject infected with SARS-CoV-2.

Viral load may be detected and/or quantified using any suitable technique, for example, by reverse transcription-polymerase chain reaction (RT-PCR) tests, branched DNA (bDNA) tests, and nucleic acid sequence-based amplification (NASBA) tests, qPCR may be used, e.g. as described in Fajnzylber, J. et al., Nat Commun 11 , 5493 (2020).

Severity of infection with SARS-CoV-2 may be assessed using markers of inflammation, such as epidermal growth factor, Eotaxin, fibroblast growth factor-basic, granulocyte colony-stimuating factor (CSF), granulocyte-macrophage CSF, hepatocyte growth factor, IFN-a, IFN-y, IL-1a, IL-1 p, IL-1 RA, IL-2, IL-2R, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40/p70) IL-13, IL-15, IL-17A, IL-17F, IL-22, IP-10, MCP-1 , MIG, MIP-1a, MIP-10, RANTES, tumor necrosis factor-a, and/or vascular endothelial growth factor, using any suitable technique, such as a Luminex xMAP assay (ThermoFisher).

Chimeric

The present disclosure also provides Chimeric Antigen Receptors (CARs) comprising the antigenbinding polypeptides or polypeptides of the present disclosure.

CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region. An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker.

The CAR of the present disclosure comprises an antigen-binding region which comprises or consists of the antigen-binding molecule of the present disclosure, or which comprises or consists of a polypeptide according to the present disclosure. The cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell. In some embodiments, the CAR comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CD3- , CD4, CD8 or CD28. As used herein, a region which is “derived from” a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.

The signalling region of a CAR allows for activation of the T cell. The CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell. Signalling regions comprising sequences of other ITAM-containing proteins such as FcyRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1):182-187). Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27. In some cases CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co- stimulatory molecule. In some embodiments, the CAR of the present disclosure comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, 0X40, 4-1 BB, ICOS and CD27.

An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Hinge regions may be derived from lgG1 . In some embodiments, the CAR of the present disclosure comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of lgG1 .

Also provided is a cell comprising a CAR according to the present disclosure. The CAR according to the present disclosure may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR- NK cells. Engineering of CARs into immune cells may be performed during culture, in vitro.

The antigen-binding region of the CAR of the present disclosure may be provided with any suitable format, e.g. scFv, scFab, etc. Nucleic acids and vectors

The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigenbinding molecule, polypeptide or CAR according to the present disclosure. In some embodiments the nucleic acid(s) comprise or consist of DNA and/or RNA.

The present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure.

Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

The nucleotide sequence may be contained in a vector, e.g. an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).

Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian expression vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. Constituent polypeptides of an antigen-binding molecule according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.

In some embodiments, a nucleic acid according to the present disclosure comprises or consists of a nucleotide sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100.

In some embodiments, a vector according to the present disclosure comprises the sequence features shown in Figure 34.

In some embodiments, a vector according to the present disclosure comprises or consists of a nucleotide sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:101.

Cells comprisinq/expressinq the antiqen-bindinq molecules and polypeptides

The present disclosure also provides a cell comprising or expressing an antigen-binding molecule, polypeptide or CAR according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure.

The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).

In some embodiments, the cell is, or is derived from, a cell type commonly used for the expression of polypeptides for use in therapy in humans. Exemplary cells are described e.g. in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include e.g. CHO, HEK 293, PER.C6, NS0 and BHK cells. In preferred embodiments, the cell is, or is derived from, a CHO cell.

The present disclosure also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction). The present disclosure also provides a method for producing a cell expressing/comprising an antigen-binding molecule, polypeptide or CAR according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.

The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.

The present disclosure also provides the cell line designated MCB-115-05, deposited 5 November 2020 as ATCC patent deposit number PTA-126858.

Producinq the antiqen-bindinq molecules and polypeptides

Antigen-binding molecules and polypeptides according to the present disclosure may be prepared according to methods for the production of polypeptides known to the skilled person.

Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides/polypeptides can by synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

Alternatively, antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451- 3461 , both of which are hereby incorporated by reference in their entirety.

In some cases, the antigen-binding molecule of the present disclosure are comprised of more than one polypeptide chain. In such cases, production of the antigen-binding molecules may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding molecule.

For recombinant production according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. a cell described hereinabove.

In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.

Production may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

Following culturing the cells that express the antigen-binding molecule/polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.

It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.

Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation.

Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

The present disclosure also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein.

The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral ortransdermal routes of administration which may include injection or infusion.

Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

In some embodiments the composition is formulated for injection or infusion, e.g. into a blood vessel ortissue/organ of interest.

The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and/or mixing antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent. For example, a further aspect the present disclosure provides a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a disease caused by SARS-CoV-2 infection), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

In aspects and embodiments of the present disclosure, the antigen-binding molecule may be provided in a composition comprising particular chemical constituents in specified concentrations/proportions.

In some embodiments, the antigen-binding molecule is provided in a buffer. As used herein, a "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. A buffer of the present disclosure preferably has a pH in the range from about 4.5 to about 7.0, preferably from about 5.0 to about 6.5. Examples of buffers that will control the pH in this range include acetate, histidine, histidine-arginine, histidine-methionine and other organic acid buffers.

In some embodiments, the composition comprising the antigen-binding molecule has a pH of 4.0 to 7.0, e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2. In some embodiments, the composition has a pH of - 5.2. In some embodiments, the composition has a pH of - 6.0.

In some embodiments, the antigen-binding molecule is provided in an acetate buffer, i.e. a buffer comprising acetate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising acetate at a final concentration of 1 mM to 100 mM acetate, e.g. one of 2 mM to 20 mM, 5 mM to 15 mM, 6 to 14 mM, or 8 to 12 mM. In some embodiments, the composition may comprise -10 mM acetate.

In some embodiments, the antigen-binding molecule is provided in a histidine buffer, i.e. a buffer comprising histidine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising histidine at a final concentration of 2 mM to 200 mM, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise -20 mM histidine.

In some embodiments, the antigen-binding molecule is provided in an arginine buffer, i.e. a buffer comprising arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising arginine at a final concentration of 1 mM to 100 mM arginine, e.g. one of 2 mM to 20 mM, 5 mM to 15 mM, 6 to 14 mM, or 8 to 12 mM. In some embodiments, the composition may comprise -10 mM arginine. In some embodiments, the antigen-binding molecule is provided in a histidine-arginine buffer, i.e. a buffer comprising histidine and arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising histidine at a final concentration of 2 mM to 200 mM, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM, and arginine at a final concentration of 1 mM to 100 mM arginine, e.g. one of 2 mM to 20 mM, 5 mM to 15 mM, 6 to 14 mM, or 8 to 12 mM. In some embodiments, the composition may comprise ~20 mM histidine and ~10 mM arginine.

In some embodiments, the antigen-binding molecule is provided in a composition comprising methionine. The methionine component of the composition may be provided at a final concentration of 1 mM to 50 mM methionine, e.g. one of 1 mM to 10 mM, 2.5 mM to 7.5 mM, 3 to 7 mM, or 4 to 6 mM. In some embodiments, the composition may comprise ~5 mM methionine.

In some embodiments, the antigen-binding molecule is provided in a histidine-methionine buffer, i.e. a buffer comprising histidine and methionine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising histidine at a final concentration of 2 mM to 200 mM, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM, and methionine at a final concentration of 1 mM to 50 mM methionine, e.g. one of 1 mM to 10 mM, 2.5 mM to 7.5 mM, 3 to 7 mM, or 4 to 6 mM. In some embodiments, the composition may comprise ~20 mM histidine and ~5 mM methionine.

In some embodiments, the composition comprising the antigen-binding molecule comprises an isotonicity agent. Isotonicity agents may be used to provide isotonic formulations. Examples of isotonicity agents include e.g. salts (e.g. sodium chloride, potassium chloride) and sugars (e.g. sucrose, glucose, trehalose).

In some embodiments, the antigen-binding molecule is provided in a composition comprising sucrose. The sucrose component of the composition may be provided at a final concentration (in weight by volume) of 2% to 20%, e.g. one of 5% to 15%, 6% to 14%, or 7% to 12%. In some embodiments, the composition may comprise ~8% (w/v) sucrose.

In some embodiments, the composition comprising the antigen-binding molecule comprises a surfactant. As used herein, a "surfactant" refers to an agent which lowers interfacial tension. The surfactant is preferably a non-ionic surfactant. Examples of surfactants include polysorbate (polysorbate-20, polysorbate-80), poloxamer (poloxamer-188) and Triton X-100. The surfactant is preferably present in the composition in the range from about 0.001 % (w/v) to about 0.5% (w/v).

In some embodiments, the antigen-binding molecule is provided in a composition comprising polysorbate-80. The polysorbate-80 component of the composition may be provided at a final concentration (in weight by volume) of 0.001 % to 0.1 %, e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise ~0.01 % (w/v) polysorbate-80. In some embodiments, the composition may comprise ~0.02% (w/v) polysorbate-80.

In some embodiments, the antigen-binding molecule is provided in a composition comprising:

1 mM to 100 mM (e.g. one of 2 mM to 20 mM, 5 mM to 15 mM, 6 to 14 mM, or 8 to 12 mM) acetate, more preferably ~10 mM acetate;

2% to 20% (e.g. one of 5% to 15%, 6% to 14%, or 7% to 12%) sucrose (w/v), more preferably ~9% (w/v) sucrose;

0.001 % to 0.1 % (e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w/v), more preferably ~0.01 % (w/v) polysorbate-80; and pH 4.0 to 7.0 (e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH ~5.2.

2 mM to 200 mM (e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably ~20 mM histidine;

2% to 20% (e.g. one of 5% to 15%, 6% to 14%, or 7% to 12%) sucrose (w/v), more preferably ~8% (w/v) sucrose;

0.001 % to 0.1 % (e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w/v), more preferably ~0.02% (w/v) polysorbate-80; and pH 4.0 to 7.0 (e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH ~5.2.

1 mM to 50 mM (e.g. one of 1 mM to 10 mM, 2.5 mM to 7.5 mM, 3 to 7 mM, or 4 to 6 mM) methionine, more preferably ~5 mM methionine; and

0.001 % to 0.1 % (e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w/v), more preferably ~0.02% (w/v) polysorbate-80; pH 4.0 to 7.0 (e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH ~5.2.

2 mM to 200 mM (e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably ~20 mM histidine; 2% to 20% (e.g. one of 5% to 15%, 6% to 14%, or 7% to 12%) sucrose (w/v), more preferably ~8% (w/v) sucrose;

0.001% to 0.1% (e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w/v), more preferably ~0.02% (w/v) polysorbate-80; and pH 4.0 to 7.0 (e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH ~6.0.

1 mM to 100 mM (e.g. one of 2 mM to 20 mM, 5 mM to 15 mM, 6 to 14 mM, or 8 to 12 mM) arginine, more preferably ~10 mM arginine;

0.001% to 0.1% (e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w/v), more preferably ~0.02% (w/v) polysorbate-80; and pH 4.0 to 7.0 (e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH ~5.2.

The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.

The present disclosure provides an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

The methods may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition. The methods may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments the methods may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments the methods may prevent development of the disease/condition a later stage.

It will be appreciated that the articles of the present disclosure may be used for the treatment/prevention of any disease/condition that would derived therapeutic or prophylactic benefit from a reduction in the level of SARS-CoV-2, or a reduction in the number of cells infected with SARS-CoV-2. For example, the disease/condition may be a disease/condition in which SARS-CoV-2 infection is pathologically implicated, e.g. a disease/condition for which SARS-CoV-2 infection is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which infection with SARS-CoV-2, is a risk factor for the onset, development or progression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition caused by SARS-CoV-2 infection, e.g. COVID-19. In some embodiments, the disease/condition to be treated/prevented is caused by infection with the SARS- CoV-2 variant B.1 .617.1 . In some embodiments, the disease/condition to be treated/prevented is caused by infection with the SARS-CoV-2 variant B.1 .617.2.

The clinical features of COVID-19 are described in Lechien et al., Journal of Internal Medicine (2020) 288(3): 335-344, International Severe Acute Respiratory and Emerging Infections Consortium (ISARIC). COVID-19 Report: 19 May 2020: ISARIC; 2020 and Docherty et al., BMJ (2020) 369:m1985, which are hereby incorporated by reference in their entirety. Common symptoms include cough, fever, headache, dyspnoea, anosmia, pharyngitis, nasal obstruction, rhinorrhoea, asthenia, myalgia, joint pain, gustatory dysfunction, abdominal pain, vomiting, and diarrhoea. The majority patients present with mild/moderate disease, however hospitalisation is sometimes required in particularly in elderly patients and/or patients having comorbidities such as diabetes and cardiovascular disease. A major complication in COVID-19 is progression to acute respiratory distress syndrome (ARDS), which presents as dyspnoea and acute respiratory failure, with patients requiring mechanical ventilation. A proportion of infected subjects are asymptomatic.

Treatment in accordance with the methods of the present disclosure may achieve one or more of: a reduction in the level or viral load of SARS-CoV-2 in the subject or in a tissue/organ of the subject (e.g. the lungs), a reduction in the level of expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in the subject or in a tissue/organ of the subject (e.g. the lungs), inhibition of the development/progression of a disease/condition caused by SARS-CoV-2 infection (e.g. COVID-19) in the subject, a reduction in the severity of symptoms of a disease/condition caused by SARS-CoV- 2 infection (e.g. COVID-19) in the subject, inhibition of the development/progression of acute respiratory distress syndrome (ARDS) in the subject, and an increase in survival of the subject. In some embodiments, a subject may be selected for treatment described herein based on the determination of SARS-CoV-2 infection, e.g. by detection of SARS-CoV-2 in a sample obtained from the subject. In some embodiments, a subject may be selected for treatment described herein based on determination that the subject is at risk of having been infected with SARS-CoV-2. For example, the subject might have been in close contact with a subject infected with SARS-CoV-2.

Administration of the articles of the present disclosure is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorderto be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

The antigen-binding molecules may be administered at a dose of about 2 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg.

Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. The antigen-binding molecule or composition described herein and a therapeutic agent may be administered simultaneously or sequentially.

Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days). The antigen-binding molecules may be administered by intravenous infusion, e.g. over a period of 60 minutes.

In accordance with various aspects of the present disclosure, a method of treating and/or preventing a disease/condition caused by SARS-CoV-2 infection, e.g. COVID-19 may comprise one or more of the following: inhibiting infection of ACE2-expressing cells by SARS-CoV-2; reducing the level of SARS-CoV-2 in a subject infected with SARS-CoV-2 (e.g. in the lung); reducing the expression of a proinflammatory factor (e.g. IL-6, CCL2 and/or CXCL10) in a subject infected with SARS-CoV-2 (e.g. in the lung); reducing the number/proportion of cells comprising/infected with SARS-CoV-2; cell killing of cells infected with SARS-CoV-2; reducing the severity of symptoms of a disease caused by SARS-CoV-2 infection in a subject infected with SARS-CoV-2; increasing survival of a subject infected with SARS-CoV-2.

Methods of detection

The present disclosure also provides the articles of the present disclosure for use in methods for detecting SARS-CoV-2/SARS-CoV-2 spike protein, or cells comprising SARS-CoV-2/SARS-CoV-2 spike protein (e.g. cells infected with SARS-CoV-2).

The antigen-binding molecules described herein may be used in methods that involve detecting binding of the antigen-binding molecule to SARS-CoV-2 spike protein. Such methods may involve detection of the bound complex of the antigen-binding molecule and SARS-CoV-2 spike protein.

As such, a method is provided, comprising contacting a sample containing, or suspected to contain, SARS-CoV-2/SARS-CoV-2 spike protein, and detecting the formation of a complex of the antigenbinding molecule and SARS-CoV-2/SARS-CoV-2 spike protein. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell comprising SARS-CoV- 2/SARS-CoV-2 spike protein, and detecting the formation of a complex of the antigen-binding molecule and a cell comprising SARS-CoV-2/SARS-CoV-2 spike protein.

Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.

Methods comprising detecting SARS-CoV-2/SARS-CoV-2 spike protein or cells comprising SARS- CoV-2/SARS-CoV-2 spike protein include methods for diagnosing infection with SARS-CoV-2, and methods for diagnosing/prognosing disease caused by infection with SARS-CoV-2, e.g. COVID-19.

Methods of this kind may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments the method is performed in vivo.

Such methods may involve detecting or quantifying one or more of: SARS-CoV-2, SARS-CoV-2 spike protein, cells comprising SARS-CoV-2 or cells comprising SARS-CoV-2 spike protein, e.g. in a patient sample. Where the method comprises quantifying the relevant factor, the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic/prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.

Detection in a sample may be used for the purpose of evaluation of SARS-CoV-2 infection, diagnosis of a disease/condition caused by infection with SARS-CoV-2 (e.g. COVID-19), predisposition to a disease/condition, or for providing a prognosis (prognosticating) for a disease/condition, e.g. a disease/condition described herein.

A sample may be taken from any tissue or bodily fluid. The sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, the sample may be obtained or derived from a tissue or tissues which are affected by the disease/condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition).

A subject may selected for diagnostic/prognostic evaluation based on the presence of symptoms indicative of SARS-CoV-2 infection in the subject’s body or in selected cells/tissues of the subject’s body, or based on the subject being considered to be at risk of developing disease caused by SARS-CoV-2, e.g. because of exposure to SARS-CoV-2 or a subject infected with SARS-CoV-2.

The present disclosure also provides methods for selecting/stratifying a subject for treatment with a SARS-CoV-2/SARS-CoV-2 spike protein-targeted agent. In some embodiments a subject is selected for treatment/prevention in accordance with the present disclosure, or is identified as a subject which would benefit from such treatment/prevention, based on detection/quantification of SARS-CoV- 2/SARS-CoV-2 spike protein, or cells comprising SARS-CoV-2/SARS-CoV-2 spike protein, e.g. in a sample obtained from the individual.

The subject in accordance with aspects of the present disclosure may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. a disease cause by infection with SARS-CoV-2), may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In embodiments according to the present disclosure the subject is preferably a human subject. In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure is a subject having, or at risk of developing, a disease described herein (e.g. COVID-19). In embodiments according to the present disclosure, a subject may be selected for 5 treatment according to the methods based on characterisation for certain markers of such disease/condition.

Kits

In some aspects of the present disclosure a kit of parts is provided. In some embodiments the kit 10 may have at least one container having a predetermined quantity of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

In some embodiments, the kit may comprise materials for producing an antigen-binding molecule, 15 polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

The kit may provide the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions for 20 administration to a patient in order to treat or prevent a specified disease/condition, e.g. COVID-19.

Sequence identity

As used herein, “sequence identity” refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, 25 after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 30 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

35 Sequences

***

The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value.

When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated. Also, where a polypeptide-encoding nucleic acid sequence is disclosed herein equivalent polypeptide-encoding sequences as a result of degeneracy of the genetic code are also expressly contemplated. Methods described herein may preferably performed in vitro. The term “in vitro" is intended to encompass procedures performed with cells in culture whereas the term “in vivo" is intended to encompass procedures with/on intact multi-cellular organisms. Brief Description of the Figures

Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

Figure 1. Bar chart showing binding of antibodies in cell culture supernatant of cells expressing the indicated antibodies to wild-type (WT) SARS-CoV-2 spike protein, the RBD of SARS- CoV-2 spike protein (RBD), or a blank negative control (-ve), as determined by ELISA.

Figure 2. Graph showing binding of the indicated purified antibodies to wild-type (WT) SARS- CoV-2 spike protein, as determined by ELISA. Kd values (in ng/ml) for each antibody-antigen pair are indicated.

Figure 3. Graph showing neutralisation of SARS-CoV-2 infection by the indicated purified antibodies. EC50 values are shown for SC31 . Error bars indicate standard error.

Figure 4. Graph showing neutralisation of binding of SARS-CoV-2 spike protein or the RBD of SARS-CoV-2 spike protein to ACE2-expressing cells by the indicated purified antibodies. Error bars indicate standard error.

Figures 5A to 5C. Graphs and table showing the results of analysis of binding of the indicated purified antibodies to the RBD of SARS-CoV-2 spike protein or SARS-CoV-2 spike protein, as determined by ELISA. (5A) shows binding to the RBD of SARS-CoV-2 spike protein. (5B) shows binding to SARS-CoV-2 spike protein. (5C) summaries binding kinetics derived from ELISA data.

Figures 6A and 6B. Graphs showing the results of analysis of neutralisation of binding of SARS- CoV-2 spike protein or the RBD of SARS-CoV-2 spike protein to ACE2-expressing cells by the indicated purified antibodies. (6A) shows neutralisation of binding of SARS-CoV-2 spike protein to ACE2-expressing cells. (6B) shows neutralisation of binding of the RBD of SARS-CoV-2 spike protein to ACE2-expressing cells. Error bars indicate standard error.

Figure 7. Graph and table showing neutralisation of SARS-CoV-2 infection by the indicated purified antibodies. IC50 values are shown. Error bars indicate standard error.

Figures 8A and 8B. Graphs showing the results of analysis of binding of the indicated purified antibodies to cells expressing SARS-CoV-2 spike protein, or cells not expressing SARS-CoV-2 spike protein, as determined by flow cytometry. (8A) shows binding to HEK293T cells overexpressing SARS-CoV-2 spike protein. (8B) shows binding to non-transfected HEK293T cells.

Figure 9. Graph showing binding of the indicated purified antibodies to human PBMCs, as determined by flow cytometry. Figures 10A to 10E. Graphs and table showing the results of analysis of thermostability by Differential Scanning Fluorimetry. (10A) to (10D) show the first derivative of the raw data obtained in the analysis triplicate samples of (10A) antibody [1] of Example 1 , (10B) antibody [2] of Example 1 , (10C) antibody [3] of Example 1 and (10D) an antibody specific for an irrelevant antigen (assay control). (10E) summarises the melting temperatures derived from the data.

Figure 11. Table showing the results of analysis of antibody aggregation propensity of the indicated antibodies as determined by HPLC analysis.

Figure 12. Table showing the results of analysis of the isoelectric point of the indicated antibodies as determined by capillary isoelectric focussing separation analysis.

Figures 13A and 13B. Tables showing the results of analysis of charge variance as determined by cation exchange high performance liquid chromatography analysis. (13A) shows the results obtained for antibody [1] of Example 1 , and (13B) shows the results obtained for antibody [3] of Example 1 ,

Figure 14. Graph showing neutralisation of SARS-CoV-2 infection by the indicated purified antibodies or IgG purified from the serum of a convalescent COVID-19 patient.

Figure 15. Graph showing binding of antibody [1] of Example 1 to the indicated SARS-CoV-2 spike protein variants, as determined by ELISA. The relative frequencies of the SARS-CoV-2 spike protein variants are shown (neg <10, low <100, mid <1000, high >1000 occurrences). Error bars indicate standard error.

Figures 16A and 16B. Graph showing the results of analysis of viral load as determined by qRT- PCR (16A) or TCIDso (16B) in lung homogenate from SARS-CoV-2-infected mice in the K18-hACE2 mouse model, at the indicated time points.

Figures 17A to 17C. Bar charts showing the results of analysis of the level of mRNA of IL-6 (17A), CCL2 (17B) and CXCL10 (17C) in lung homogenate from SARS-CoV-2-infected mice in the K18- hACE2 mouse model, at the indicated time points. Fold change above the level detected in uninfected mice is shown, adjusted for GADPH expression. Each data point represents a single mouse.

Figures 18A and 18B. Graphs showing the results of analysis of weight (18A) and survival (18B) of uninfected mice and SARS-CoV-2-infected mice in the K18-hACE2 mouse model. Figures 19A and 19B. Graph showing the results of analysis of viral load as determined by qRT- PCR (19A) or TCIDso (19B) in lung homogenate at the indicated time points, from SARS-CoV-2- infected mice treated with the indicated quantity of antibody [1] of Example 1 or an isotype control antibody, in the K18-hACE2 mouse model.

Figures 20A to 20C. Bar charts showing the results of analysis of the level of mRNA of IL-6 (20A), CCL2 (20B) and CXCL10 (20C) in lung homogenate at the indicated time points from SARS-CoV-2- infected mice treated with the indicated quantity of antibody [1] of Example 1 or an isotype control antibody, in the K18-hACE2 mouse model. Fold change above the level detected in uninfected mice is shown, adjusted for GADPH expression. Each data point represents a single mouse.

Figures 21A and 21B. Graphs showing the results of analysis of weight (21A) and survival (21B) of SARS-CoV-2-infected mice treated with the indicated quantity of antibody [1] of Example 1 or an isotype control antibody, in the K18-hACE2 mouse model.

Figures 22A and 22B. Graph showing the results of analysis of viral load as determined by qRT- PCR (22A) or TCIDso (22B) in lung homogenate at the indicated time points, from SARS-CoV-2- infected mice treated with 20 mg/kg of antibody [1] of Example 1 administered at the indicated number of hours post-infection with SARS-CoV-2, or untreated mice, in the K18-hACE2 mouse model.

Figures 23A to 23C. Bar charts showing the results of analysis of the level of mRNA of IL-6 (23A), CCL2 (23B) and CXCL10 (23C) in lung homogenate at the indicated time points from SARS-CoV-2- infected mice treated with 20 mg/kg of antibody [1] of Example 1 administered at the indicated number of hours post-infection with SARS-CoV-2, or untreated mice, in the K18-hACE2 mouse model. Fold change above the level detected in uninfected mice is shown, adjusted for GADPH expression. Each data point represents a single mouse.

Figures 24A and 24B. Graphs showing the results of analysis of weight (24A) and survival (25B) of SARS-CoV-2-infected mice treated with 20 mg/kg of antibody [1 ] of Example 1 administered at the indicated number of hours post-infection with SARS-CoV-2, or untreated mice, in the K18-hACE2 mouse model.

Figures 25A to 25E. Bar charts showing the results of analysis of different treatments on aggregation of antibody [1] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. (25A) to (25E) show the percentage aggregation following (25A) incubation at 4°C, (25B) incubation at 40°C, (25C) incubation at 50°C, (25D) agitation, and (25E) freeze/thaw treatment, for the indicated number of days (bars for each formulation from left to right are for Day 1 , Day 2, Day 3 and Day 6). Figures 26A to 26E. Bar charts showing the results of analysis of different treatments on aggregation of antibody [3] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. (26A) to (26E) show the percentage aggregation following (26A) incubation at 4°C, (26B) incubation at 40°C, (26C) incubation at 50°C, (26D) agitation, and (26E) freeze/thaw treatment, for the indicated number of days (bars for each formulation from left to right are for Day 0, Day 1 , Day 3 and Day 6).

Figure 27. Bar chart showing the results of analysis of oxidation treatment on aggregation of antibody [1] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. Percentage aggregation following the indicated treatment, for the indicated number of days, is shown (bars for each formulation from left to right are for Non-oxidation, 0.01% oxidation, and 0.1 oxidation).

Figure 28. Bar chart showing the results of analysis of oxidation treatment on aggregation of antibody [3] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. Percentage aggregation following the indicated treatment, for the indicated number of days, is shown (bars for each formulation from left to right are for Non-oxidation, 0.01% oxidation, and 0.1 oxidation).

Figure 29. Bar chart showing the results of analysis of concentration treatment on aggregation of antibody [1] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. Percentage aggregation at 20 mg/ml, 50 mg/ml and 150 mg/ml are shown.

Figure 30. Bar chart showing the results of analysis of concentration treatment on aggregation of antibody [3] of Example 1 provided in the indicated formulations according to Example 5.1 , as determined by SE-HPLC analysis. Percentage aggregation at 20 mg/ml, 50 mg/ml and 150 mg/ml are shown.

Figures 31 A to 31 C. Graphs, tables and bar chart showing the results of analysis of binding of antibody [3] of Example 1 to the RBD of SARS-CoV-2 spike protein as determined by ELISA, after being subjected to different treatments in the indicated formulations. (31 A) and (31 B) show binding at Day 0 (DO), following incubation at 4°C for 6 days (4°C), incubation at 40°C for 6 days (40°C), incubation at 50°C for 6 days (50°C), agitation for 6 days (Agitation), or following 6 days of freeze/thaw treatment (FT), for antibody [3] of Example 1 provided in formulation F5 (31 A) and F13 (31 B). (31 C) shows EC50 values derived from the ELISA data (bars for each formulation from left to right are for DO, 4°C, 40°C, 50°C, Agitation and FT).

Figures 32A and 32B. Graph, table and bar chart showing the results of analysis of binding of antibody [3] of Example 1 to the RBD of SARS-CoV-2 spike protein as determined by ELISA, after being subjected to different treatments in the indicated formulations. (32A) shows binding in the absence of oxidation treatment, or following 24 h of oxidation using 0.01 % H2O2 (Oxi 0.01) or 0.1 % H2O2 (Oxi 0.1), for antibody [3] of Example 1 provided in formulation F5 or F13. (32B) shows EC50 values derived from the ELISA data (bars for each formulation from left to right are for no oxidation, 0.01 % oxidation and 0.1 % oxidation).

Figure 33. Graph and table showing the results of analysis of binding of antibody [3] of Example 1 to the RBD of SARS-CoV-2 spike protein as determined by ELISA, after being subjected to different treatments in the indicated formulations. Binding following concentration of antibody [3] of Example 1 to the indicated concentrations in formulation F5 or F13 is shown.

Figure 34. Schematic representation of the features of expression vector pDZ-AOD01 encoding antibody [3] of Example 1 .

Figures 35A to 35D. SC31 binds SARS-CoV-2 SP and neutralizes virus through inhibition of binding to ACE2. (35A) Neutralization with 100 TCID50 of infectious virus by SC31 lgG1 in comparison to control Ig G 1 or IgG purified from donor serum. (35B) Neutralization of 100 or 1000 TCID50 infectious virus by SC31 lgG1. Neutralization efficacy is represented as a percentage relative to uninfected and virus only controls. (35C) Binding affinity of SC31 IgG to purified WT-spike or RBD as measured by ELISA. (35D) Inhibition of WT-spike or RBD binding to cells expressing membranebound ACE2 by SC31 lgG1 at different concentrations as measured by flow cytometry. Binding is expressed as a percentage relative to no antibody or no viral protein controls. All results represent the mean of three independent replicates with bars showing standard error.

Figures 36A to 36C. SC31 binding to SP variants identifies its ACE2 interface epitope. (36A) Binding affinity of SC31 as determined by ELISA to purified wild-type spike and spike mutants that either do not affect SC31 or ACE2 binding, affect SC31 but not ACE2 binding, or affect both SC31 and ACE2 binding. Results are the mean of three independent replicates and are represented as a percentage of maximal absorbance against wild-type spike at the highest antibody concentration. (36B). Binding affinity of purified wild-type and mutant spike protein to hACE2-expressing CHO cells based on fluorescence intensity measured by flow cytometry. Results are the mean of three independent replicates with bars showing the standard error and are represented relative to wildtype spike binding to ACE2. Statistical significance was determined vs. wild-type for each mutant by one-way ANOVA: ** p<0.01 , *** p<0.001 , **** p<0.0001 . (36C). Location of single amino acid mutations on a crystal structure of RBD showing the interaction of the RBM with ACE2 N-terminal helix.

Figure 37A to 37G. SC31 requires Fc effector functions for optimal therapeutic benefit. (37A)

Activation of ADCC signaling by SC31 or null-binding LALA variant at various concentrations incubated with FcyRllla-expressing reporter cell line and spike or mock transfected HEK293, as determined by luciferase expression. Statistical significance in comparison to the highest SC31 concentration of 4ug/ml was determined by one-way ANOVA. (37B). Specific binding of SC31 to spike transfected HEK293 cells in comparison to mock transfected cells or fluorophore stain only (Ctrl). (37C) Overview of therapeutic study design with SC31 lgG1 and LALA variant antibodies. Antibodies were dosed at 20mg/kg 6hpi and 5 mice sacrificed at 3dpi to ascertain lung viral and cytokine load as well as antibody serum titres with the remainder monitored for weight and survival. (37D) Lung viral load of lgG1 LALA-treated or untreated mice at 3dpi as measured by genome copies (left) or infectious virus (right). The limit of detection (LOD) is indicated by the dotted line. (37E) Disease progression in infected mice as indicated by weight loss (left) or survival (right). (37F) Lung cytokine mRNA expression determined by qRT-PCR and represented as fold-change over uninfected mice. (37G) Sera IFNy protein level determined by sandwich ELISA. Each point represents one individual mouse and all bars show standard error.

Figures 38A to 38C. SC31 does not induce ADE. (38A and 38B) Lack of SARS-CoV-2 pseudovirus infection co-incubated with SC31 or LALA variant in FcyRllla expressing cell lines THP-1 (38A) and Raji (38B) in comparison to ACE2-expressing CHO cell line based as determined by luciferase reporter gene expression. (38C) Retention of SC31 binding affinity for WT-spike between pH4.5-7.0 as measured by ELISA. All results represent the mean of three or four independent replicates with bars showing standard error.

Figures 39A to 39E. Dose dependent therapeutic benefit of SC31 . (39A) Overview of therapeutic study design with different SC31 lgG1 doses or isotype control at 20mg/kg. Antibodies were dosed at 6hpi and 5 mice sacrificed at 3dpi to ascertain lung viral and cytokine load as well as antibody serum titres with the remainder monitored for weight and survival. (39B) Disease progression in infected mice as indicated by weight loss (left) or survival (right). (39C-39D) Lung viral load of antibody treated mice at 3dpi as measured by genome copies (39C) or infectious virus (39D). The limit of detection (LOD) is indicated by the dotted line. (39E) Lung cytokine mRNA expression determined by qRT-PCR and represented as fold-change over uninfected mice. Each point represents one individual mouse and all bars show standard error.

Figures 40A to 40E. Efficacious dose window for SC31 is before viral peak. (40A) Overview of therapeutic study design with SC31 IgG 1 doses at 20mg/kg at different timepoints. 5 mice were sacrificed at 3dpi to ascertain lung viral and cytokine load as well as antibody serum titres with the remainder monitored for weight and survival. (40B) Disease progression in infected mice as indicated by weight loss (left) or survival (right). Lung viral load of antibody treated mice at 3dpi as measured by genome copies (40C) or infectious virus (40D). The limit of detection (LOD) is indicated by the dotted line. (40E) Lung cytokine mRNA expression determined by qRT-PCR and represented as fold-change over uninfected mice. Each point represents one individual mouse and all bars show standard error. Figure 41. Discovery of SC31 from single B cells. Dot plot showing gating strategy for sorting spikebinding IgG B cells from patient PBMCs samples HJ57 and CSY63.

Figures 42A and 42B. Determination of SC31 binding to Spike variants. (42A) Binding affinity of SC31 to purified wild-type spike and spike mutants as determined by ELISA. Results are the mean of three independent replicates and are represented as a percentage of maximal absorbance against wild-type spike at the highest antibody concentration. (42B) Binding affinity of purified wildtype and mutant spike protein to hACE2-expressing CHO cells as determined by fluorescence intensity with flow cytometry. Results are the mean of three independent replicates with bars showing the standard error and are represented relative to wild-type spike binding to ACE2. Only mutations within the RBD region were tested.

Figures 43A to 43C. Establishment of K-18 human ACE2 transgenic mouse SARS-CoV-2 infection model. (43A) Disease progression in K18 mice as shown by weight loss (left) and survival (right). (43B) Kinetics of viral infection in K18 mice with lung viral load based on genome copies (left) and infectious disease (right). The dotted line indicates the limit of detection (LOD). (43C). Kinetics of the cytokine response in the lung as measured by mRNA expression of pro-inflammatory cytokines IFNp, TNF, IL1 b, IL6 and chemokines CCL2, CXCL10 represented as fold-change over uninfected mice. Each point represents one individual mouse with the mean indicated by the horizontal lines or bars. Statistical significance between viral load on adjacent days was determined using Student’s t- test.

Figure 44. Table showing Charge Variant Analysis (CEX-HPLC) and Isoelectric Point (clEF) of SC31 parental and SC31 engineered demonstrating the improvement in developability.

Figure 45. Graph showing mean (SE) serum concentrations of SC31 versus time at the four dose levels in healthy human adults.

Examples

In the following Examples, the inventors describe the identification and characterisation of antibodies capable of binding to SARS-CoV-2 spike protein, and formulation and cell line development for an exemplary SARS-CoV-2 spike protein-specific antibody.

Example 1 : Antibodies specific for SARS-CoV-2 spike protein

The inventors identified antibodies capable of binding to SARS-CoV-2 spike protein: SC1 , SC11 , and SC31WT.

The sequences of SC1 , SC11 , and SC31WT were analysed for sequence liabilities that would be undesirable in terms of commercial antibody production (e.g. sequences presenting a risk of aggregation of the antibody, deamidation, isomerisation, oxidation, post-translational modification) and/or in relation to their use in therapy in humans (e.g. immunogenic sequences).

Engineered variants were designed having optimised sequences: SC31 GS, SC31 GSeng, SC1 GS, SC11 GS, SC11 GSeng1 and SC11 GSeng2.

The amino acid and nucleotide sequences of the VH and VL domain sequences of the antibodies are shown below.

The antibodies were produced in human lgG1 format.

Briefly, DNA sequences encoding the heavy chain variable region sequences were cloned (in frame) into expression vectors encoding the constant region sequences of human IgG 1 , and DNA sequences encoding the light chain variable region sequences were cloned (in frame) into expression vectors encoding the constant region sequences of human kappa light chain.

The amino acid sequences of the heavy and light chains of the antibodies are shown in the table below. Antibodies formed of the heavy and light chains were produced by expression of vectors encoding the heavy and light chains from mammalian cells.

Example 2: Initial characterisation of SARS-CoV-2 spike protein-binding antibodies

Antibody variable heavy and light chain sequences of antibodies [1], [4] and [6] of Example 1 were cloned into pCMV-promoter driven expression plasmids containing the appropriate human lgG1 heavy, kappa or lambda light chain constant regions as well as a leader sequence for secretory expression. Purified plasmids were transfected into HEK293 cells in suspension cell culture in F17 medium (ThemoFisher) at a concentration of 1 .Omg DNA/L of culture using branched polyethylenimine (Sigma-Aldrich) at 3:1 w/w ratio of PEI:DNA. Culture supernatant was harvested after 7 days, either used directly in experiments or monoclonal antibodies were purified from the cell culture supernatants by HPLC using or MabSelect SuRe Columns (Cytiva).

2.1 Analysis of binding to SARS-CoV-2 spike protein

Cell culture supernatant containing antibodies [1], [4] or [6] of Example 1 was used in ELISA assays for evaluating their ability to bind to SARS-CoV-2 spike protein and the RBD of SARS-CoV-2 spike protein.

Briefly, 2 pg/ml purified SARS-CoV-2 spike protein, or the RBD of SARS-CoV-2 spike protein was coated in binding buffer (100 mM Tris-HCI, 1 mM EDTA, 150 mM NaCI, pH 8.0) onto Streptactin XT 96-well ELISA plates (IBA GmBH) for 2 h at room temperature. Plates were washed with PBS and 100 pL of cell culture supernatant containing expressed antibodies was diluted into 2% BSA/PBS blocking solution and incubated for 1 h at room temperature before washing thrice with PBS/0.05%Tween. Antibody binding was detected using 1 :5000 anti-huIgG Fc-HRP conjugated secondary antibody (ThermoFisher) diluted in blocking solution incubated for 1 h at room temperature. Plates were then washed thrice with PBS/0.05% Tween and once with PBS before signal development with TMB solution (ThermoFisher), which was stopped using 2M sulphuric acid. All assay volumes were at 100 pl/well.

The results are shown in Figure 1 . Antibodies [1], [4] and [6] were confirmed to bind to SARS-CoV-2 spike protein. Antibody [1] was moreover shown to bind to the RBD of SARS-CoV-2 spike protein.

In further ELISA experiments, preparations of antibodies [1], [4] or [6] of Example 1 purified from cell culture supernatants were evaluated fortheir affinity for binding to SARS-CoV-2 spike protein.

The experiments were performed essentially as described above, using the indicated concentrations of the purified antibodies, in triplicate. ELISA graphs were plotted and antibody affinity determination was performed using PRISM software. Kd values in ng/ml were calculated. The results are shown in Figure 2.

2.2 Analysis of ability to inhibit SARS-CoV-2 infection in vitro

Purified antibodies [1], [4] or [6] of Example 1 were next evaluated for their ability to neutralise infection of ACE2-expressing cells in vitro.

Microneutralization assays were performed using VeroE6 cells with OTCIDso of SARS-CoV-2. An isotype-matched antibody specific for an irrelevant antigen was included as a negative control. Briefly, SARS-CoV-2 virus obtained from a patient nasal swab was cultured in VeroE6 cells, and supernatant harvested on observation of 90% CPE. The antibodies were incubated at indicated concentrations with 100 TCID50 of virus and 2x10⁴ VeroE6 cells in 100 pl of culture media (MEM/2% FCS) in 96-well flat bottom plates, and incubated for 72 h at 37°C in 5% CO2. Experiments were performed in duplicate. Neutralization was measured using Viral Toxglo reagent (Promega) to determine percentage cell survival relative to “no virus” (i.e. cells only) and “virus only” (i.e. no antibody) control conditions.

The results are shown in Figure 3. Antibody [1] was shown to potently neutralise infection of cells by SARS-CoV-2.

2.3 Analysis of ability to inhibit SARS-CoV-2 spike protein interaction with ACE2

Antibodies [1] and [10] were next analysed for their ability to inhibit interaction between SARS-CoV-2 spike protein and ACE2-expressing cells in vitro.

Briefly, 20mM Twin-strep-tagged SARS-CoV-2 spike protein or RBD of SARS-CoV-2 spike protein was incubated with antibody for 1 h in 50 pl FACS buffer (1 X PBS, 0.5% BSA and 2mM EDTA) at 4°C. 5x10⁴ huACE2-expressing CHO cells in 50 pl FACS buffer were added, and incubated for a further 1 h at 4°C. Experiments were performed in duplicate. Cells centrifuged at 300 x g for 5 min, washed twice with PBS and then stained with 1 :50 Alexa488-conjugated anti-Strep-Tag II antibody (StrepMAb-lmmuno, IBA GmBH) for 30 min in 50 pl FACS buffer. Cells were finally washed once with 1x PBS, stained with 1 :100 propidium iodide (PI) and analysed on a BD FACS Canto II. Plnegative cells were gated and binding was measured by analysis of 488 channel fluorescence intensity. Inhibition of binding was calculated as a percentage relative to unlabeled cells (0%) or labelled cells in the absence of antibody (100%).

The results are shown in Figure 4. Antibody [1] was shown to inhibit binding of SARS-CoV-2 spike protein or the RBD of SARS-CoV-2 spike protein to ACE2-expressing cells. Example 3: Further characterisation of SC31WT, SC31GS and SC31GSenq

Antibodies [1], [2] and [3] of Example 1 were produced by co-transfection of HEK 293 cells with vectors encoding the antibody heavy and light chains, and purification of the expressed antibodies from cell culture supernatants.

3. 1 Analysis of binding to SARS-CoV-2 spike protein

Antibodies [1], [2] and [3] of Example 1 were analysed for their ability to bind to SARS-CoV-2 spike protein and the RBD of SARS-CoV-2 spike protein by ELISA.

ELISAs were performed as described in Example 2.1 above.

The results are shown in Figures 5A to 5C. Antibodies [1], [2] and [3] were confirmed to bind to SARS-CoV-2 spike protein and also to the RBD of SARS-CoV-2 spike protein.

3.2 Analysis of ability to inhibit SARS-CoV-2 spike protein interaction with ACE2

Antibodies [1], [2] and [3] were analysed for their ability to inhibit interaction between SARS-CoV-2 spike protein or the RBD of SARS-CoV-2 spike protein with ACE2-expressing CHO cells in vitro.

Antibody CR3022 (described e.g. in ter Meulen et al., PLoS Med. (2006) 3(7): e237) which is known to bind to the RBD of SARS-CoV-2 was included as a control.

Experiments were performed as described in Example 2.3 above.

The results are shown in Figures 6A and 6B. Antibodies [1], [2] and [3] were shown to inhibit binding of SARS-CoV-2 spike protein (Figure 6A) or the RBD of SARS-CoV-2 spike protein (Figure 6B) to ACE2-expressing cells.

3.3 Analysis of ability to inhibit SARS-CoV-2 infection in vitro

Antibodies [1], [2] and [3] were analysed for their ability to neutralise infection of ACE2-expressing cells in vitro.

Microneutralization assays were performed as described in Example 2.2 above. IC50 values were derived from the neutralisation data.

The results are shown in Figure 7. Antibodies [1], [2] and [3] were shown to potently neutralise infection of cells by SARS-CoV-2.

3.4 Analysis of ability to bind to cells expressing SARS-CoV-2 spike protein

Antibodies [1], [2] and [3] were analysed by flow cytometry for their ability to bind to cells overexpressing SARS-CoV-2 spike protein. An antibody known to bind to SARS-CoV-2 spike protein was included as a positive control, and an isotype-matched antibody specific for an irrelevant antigen was included as a negative control.

HEK293T cells were transfected with pTT5 expression vector encoding SARS-CoV-2 spike protein using Lipofecatmine 2000 (Thermo Scientific, #1668-01) in accordance with the manufacturer’s instructions.

Non-transfected HEK293T cells or HEK293T cells overexpressing SARS-CoV-2 spike protein were washed twice with PBS and resuspended in FACS buffer (1 X PBS, 0.5% BSA and 2mM EDTA). 50,000 cells/well were seeded in wells of a 96-well plate. Cells were resuspended in serial dilutions of the antibodies, and incubated for 1 h at 4°C. Cells were subsequently washed 3 times with FACS buffer and resuspended in a 1 :200 dilution of Alexa Fluor 488-conjugated secondary goat antiHuman IgG (H+L) (Cross-Adsorbed; #A11013) for 1 h at 4°C. Cells were subsequently washed 3 times with FACS buffer and resuspended in resuspension buffer (1 X PBS, 0.5% BSA and 2mM EDTA + DAPI (1 :200 dilution)). Cells were then analysed by flow cytometry using MACSQuantX.

The results are shown in Figures 8A and 8B. Antibodies [1], [2] and [3] displayed dose-dependent binding to cells overexpressing SARS-CoV-2 spike protein, but did not display any binding to nontransfected HEK 293T cells.

3.5 Analysis of binding to PBMCs

Antibodies [1], [2] and [3] were analysed by flow cytometry to determine whether they bind to human PBMCs. A CD47-specific antibody known to bind to human PBMCs was included as a positive control, and an isotype-matched antibody specific for an irrelevant antigen was included as a negative control.

Human PBMCs were washed twice with PBS and resuspended in FACS buffer (1 X PBS, 0.5% BSA and 2mM EDTA). Cells were incubated with blocking solution (Human TruStain FcX (#422302) at room temperature for 15 min. 50,000 cells/well were then seeded in wells of a 96-well plate. Cells were resuspended in serial dilutions of the antibodies, and incubated for 1 h at 4°C. Cells were subsequently washed 3 times with FACS buffer and resuspended in a 1 :200 dilution of Alexa Fluor 488-conjugated F(ab')2-Goat anti-Human IgG Fc (Thermo Fisher #H10120) for 1 h at 4°C. Cells were subsequently washed 3 times with FACS buffer and resuspended in resuspension buffer (1 X PBS, 0.5% BSA and 2mM EDTA + DAPI (1 :200 dilution)). Cells were then analysed by flow cytometry using MACSQuantX.

The results are shown in Figure 9. Antibodies [1], [2] and [3] did not display any non-specific binding to human PBMCs. 3.6

Thermostability of antibodies [1], [2] and [3] was analysed by Differential Scanning Fluorimetry. An antibody specific for another antigen was included in the analysis as an assay control.

Briefly, antibodies were diluted to 0.2 mg/ml in PBS and 2.5x SYPRO orange dye (Thermo Fisher). The fluorescence of SYPRO orange was measured in a melting curve experiment performed using a thermocycler (ABI 7500fast). Temperature was increased from 25°C to 95°C with a ramp rate of 1 .2% corresponding to 1 °C/min. A melting curve was then plotted and melting temperatures (T_m) were determined from the first derivative plot of the melting curve.

The results are shown in Figures 10A to 10E. Antibodies [1], [2] and [3] were found to have a melting temperature greater than 64°C. Thus, engineering of SC31 to SC31GSeng did not significantly affect melting temperature.

3.7 Analysis of aggregation propensity

The aggregation propensity of antibodies [1], [2] and [3] was analysed by size exclusion chromatography (SEC).

Antibody purity was analyzed by size exclusion chromatography (SEC) using XBridge Protein BEH SEC columns (200A, 3.5 pm, 7.8 mm X 300 mm; Cat#186007640, Waters) on a AKTA Explorer liquid chromatography system (GE Healthcare, UK). Antibody samples were injected to SEC columns at a concentration of 1 mg/ml (in 0.22 pM filtered 1x ETF PBS), and mobile phase buffer A (0.22 pM filtered 200mM NaCI in 100mM potassium phosphate, pH 6.79) was pumped to the column at a flow rate of 1 ml/min. The run time was 15 min per injection.

The results are summarised in the table of Figure 11 . Antibodies [1], [2] and [3] were found to have minimal aggregation propensity.

3.8 Analysis of isoelectric point

The isoelectric point of antibodies [1], [2] and [3] was analysed by capillary isoelectric focussing (clEF) separation.

Briefly, the antibodies were diluted in Tris buffer (20 mM Tris, pH 8.0), and subjected to clEF separation using Pharmalyte 3-10 and the PA800 plus platform (Beckman), according to the manufacturer’s instructions. Details of the equipment and reagents used are as follows:

Instrument: PA 800 plus Pharmaceutical Analysis System, SN: 306332 (Beckman Coulter, #A66528)

Detector: PA 800 Capillary Electrophoresis series with UV/Vis detection (Beckman Coulter, #A144733)

Capillary: Bare, fused-silica capillaries of 50 pm ID x 20 cm (Beckman Coulter, #338451) clEF kit: Advanced clEF Starter Kit (Beckman Coulter, #A80976) and pl Marker Kit (Part Number, #A58481)

Working sample concentration: 5.0 mg/ml

Replicates: 3

The results are summarised in the table of Figure 12. Antibodies [2] and [3] were found to have a pl greater than 7.8. Antibodies [2] and [3] were found to have a higher pl than antibody [1], which is advantageous for antibody purification.

3.9 Analysis of charge variance

The charge variance of antibodies [1] and [3] was analysed by cation exchange high performance liquid chromatography (CEX-HPLC).

Briefly, the antibodies were subjected to HPLC analysis using YMC BioPro SP-F columns, according to the manufacturer’s instructions. Details of the reagents ad methods used are as follows:

10x CX-1 pH Gradient Buffer A (pH 5.6) (Thermo Scientific, #085346) 10x CX-1 pH Gradient Buffer B (pH 10.2) (Thermo Scientific, #085348). Sample diluent: 10x CX-1 pH Gradient Buffer A (pH 5.6)

Column: YMC BioPro SP-F, 4.6 x 100 mm, 5 pm (YMC Co. Ltd., #SF00S05-1046WP) Antibody concentration in sample: 1 mg/ml Load: 35 pg Flow rate: 0.8 ml/min

The results are shown in Figures 13A and 13B. Antibody [3] was found to have an improved charge variance profile as compared to antibody [1], indicative of improved stability, and decreased risk of low purification yields during antibody purification by ion exchange chromatography.

3.10 Analysis of interaction with lysozyme

Antibodies [2] and [3] were analysed by biolayer interferometry (BLI) for the ability to interact with lysozyme, using the Octet QK384 system (ForteBio).

All measurements were performed at 25°C with agitation at 1000 rpm. Anti-Penta-HIS (HIS1 K) coated biosensor tips (Cat#: 18-512; ForteBio, USA) were used to capture His-tagged human lysozyme 2 protein (Sino Biological, Cat#: 13726-H08B; immobilisation concentration: 4.86 pg/ml in PBS). Binding by different concentrations of antibody in PBS (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM) was evaluated as follows: baseline: 60s; loading: 120s; baseline 2: 60s; association: 120 s; dissociation: 120s; regeneration: 40 s.

Antibodies [2] and [3] did not show any significant non-specific interaction with lysozyme. Responses detected by BLI analysis were less than 0.03 nm, and within baseline noise. Example 4: Further characterisation of SC31 WT

Mammalian cells were co-transfected with vectors encoding antibody heavy and light chains for the production of antibody [1] of Example 1. 7 days post-transfection, cell culture supernatants were collected and monoclonal antibodies were purified on MabSure Select columns.

4.1 Analysis of ability to inhibit SARS-Co V-2 infection in vitro

The ability of antibody [1] to neutralise infection of ACE2-expressing cells in vitro was compared to that of serum obtained from a convalescent COVID-19 patient.

Microneutralization assays were performed as described in Example 2.2 above.

The results are shown in Figure 14. Antibody [1] was found to be ~3 times more effective at neutralising infection of ACE2-expressing cells by SARS-CoV-2 than convalescent serum.

4.2 Analysis of binding to SARS-CoV-2 spike protein variants

Antibody [1] was evaluated for its ability to bind to SARS-CoV-2 spike protein variants by ELISA.

36 spike protein variants were generated from a construct encoding wildtype SARS-CoV-2 spike protein by site-directed mutagenesis.

The indicated purified SARS-CoV-2 spike protein variants were coated in binding buffer (100 mM Tris-HCI, 1 mM EDTA, 150 mM NaCI, pH 8.0) at a concentration of 2 pg/mL onto Streptactin XT 96- well ELISA plates (IBA GmBH) for 2 h at room temperature. Plates were washed with PBS and the indicated concentrations of antibody [1] was diluted into 2% BSA/PBS blocking solution and incubated for 1 h at room temperature before washing thrice with PBS/0.05%Tween. Antibody binding was detected using 1 :5000 anti-huIgG Fc-HRP conjugated secondary antibody (ThermoFisher) diluted in blocking solution incubated for 1 h at room temperature. Plates were then washed thrice with PBS/0.05% Tween and once with PBS before signal development with TMB solution (ThermoFisher), which was stopped using 2M sulphuric acid. All assay volumes were at 100 pl/well.

The results are shown in Figure 15. Antibody [1] was found to retain strong binding to a large number of the known SARS-CoV-2 spike protein variants, including the prevalent D614G, N439K and S477N mutants.

The ability of antibody [1] to bind to additional spike protein variants was tested. Antibody [1] was found to retain strong binding to SARS-CoV-2 spike proteins comprising mutations K417N, N440K, N448Y, Y449H, L452M, L452R, S459Y, A475S, S477R, T478K, T478R, G485R, F486L, and/or G496S, as well as to the spike proteins of variants B.1 .617.1 and B.1 .617.2. 4.3 Analysis of therapeutic efficacy in vivo

The effect of treatment with antibody [1] was evaluated in a mouse model of disease caused by SARS-CoV-2 infection.

Previous investigations have shown that SARS-CoV is able to establish robust infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), resulting in death of mice by day 8 post-infection (12, 13). Six to eleven months old transgenic hACE2 mice have recently been used in an equivalent model for SARS-CoV-2 infection (14). Infected mice display weight loss by day 5 post-infection and subsequently recovered by day 14 post-infection. Lung viral load peaks at around day 3 post-infection and cleared by day 7 post-infection. Four to six month old hACE2-expressing mice have also been used to show close contact transmission of the SARS-CoV- 2, about 50% of uninfected mice housed with infected mice developed antibodies against SARS- CoV2 (15).

The transgenic human ACE2 mouse model (K18-hACE2) has been shown to support robust high level SARS-CoV-2 infection which can be lethal (16). This model has also been shown to present with extensive lung damage caused by lymphocyte infiltration into the lungs of infected mice, which is correlated with elevated mRNA expression of pro-inflammatory cytokines/chemokines such as IFNp, TNFa, IL1 b, IL6, CXCL10 and CCL2 (16). Cytokines such as IL6 and TNFa have also been shown to have clinical correlation with severe inflammation and disease in human COVID-19 infection (17).

K-18 Human ACE2 transgenic mice were supplied by the Jackson Laboratory. Female mice between 7 and 12 weeks old were used in the studies experiments. To determine the day of peak viral load, following acclimatization of the mice to the isocages, groups of mice were anesthetized individually with 3% isoflurane using the precision vaporizer and infected intranasally (I.N.) with 50pl of 1 .24x10⁴ TCIDso of SARS-CoV-2. Following infection, mice were transferred to new isocages. On the indicated days, four mice were euthanized by carbon dioxide asphyxiation and transferred to the BSC, the lungs were harvested, weighed and made to 10%w/v with viral grow medium then mashed through a disposable mesh using a plunger and aliquoted into screw cap tube and stored at -80°C, for later determination of lung viral load by qualitative real-time PCR (qRT-PCR) and cell culture to determine the tissue culture infective dose (TCID), and for analysis of cytokine/chemokine mRNA expression. Mice in survival groups were weighed on the indicated days. Any mice that show >20% weight loss or significant inactivity were euthanized humanely by carbon dioxide asphyxiation.

The results are shown in Figures 16A and 16B, Figures 17A to 17C and Figures 18A and 18B. Viral load peaked around day 3, and this was associated with an elevation in the levels of proinflammatory IL-6, CCL2 and CXCL10 in the lungs. All mice succumbed to infection by day 8. In further experiments, the effects of treatment with antibody [1] of Example 1 were investigated. K- 18 Human ACE2 transgenic mice were infected with SARS-CoV-2 as described above, anesthetized with 3% isoflurane and administered with 20, 10, 5 or 2 mg/kg of antibody [1] or 20 mg/kg of an isotype-matched antibody specific for an irrelevant antigen (as a negative control) in a total volume of 200 pl PBS by intra-peritoneal (I.P.) injection, at 6 hours post-infection. Mice were monitored and tissues were harvested and analysed as described above.

The results are shown in Figures 19A, 19B, Figures 20A to 20C and Figures 21 A and 21 B. Treatment with antibody [1] was found to reduce viral load, protect against lethality and reduce the levels of IL-6, CXCL10 and CCL2. The antibody displayed dose-dependent therapeutic effects.

In further experiments, the inventors investigated the effect of treatment with antibody [1] of Example 1 administered at different time points after SARS-CoV-2 infection. K-18 Human ACE2 transgenic mice were infected with SARS-CoV-2 as described above, anesthetized with 3% isoflurane and administered with 20 mg/kg of antibody [1] in a total volume of 200 pl PBS by intra-peritoneal (I.P.) injection at 6, 24 or 48 hours post-infection with SARS-CoV-2. Mice were monitored and tissues were harvested and analysed as described above.

The results are shown in Figures 22A, 22B, Figures 23A to 23C and Figures 24A and 24B. The greatest treatment effects were observed when antibody [1] was administered within 6 hours or 24 hours of SARS-CoV-2 infection (prior to peak viral load having been reached).

Example 5: Antibody formulation development

Formulation development was performed for antibodies [1] and [3] of Example 1 .

5.1 Buffer formulations

The antibodies were expressed in mammalian cells and purified from cell culture supernatant using protein A, followed by cation exchange chromatography and anion exchange chromatography, into a final buffer of 20 mM Tris, 49 mM NaCI, pH 8.0.

Purified antibody preparations were buffer-exchanged by overnight dialysis at 4°C into buffers having the following formulations:

| | (w/v) polysorbate 80, pH 5.2

Buffers were replaced the day after by 1 L of new buffer and dialysed for a further 2 hours. Recovered samples were then concentrated to reach a concentration of 20 mg/ml.

5.2 Treatments

Samples of the antibodies provided in the different buffer formulations were subjected to different treatments, as summarised below:

• Freeze/thaw treatment o A) 0.25 ml of 20 mg/ml sample frozen on Day 0. o B) Sample thawed at room temperature for 2 hours on Day 1 , 25 pl collected from thawed sample and maintained at 4°C until analysis. Remainder of sample refrozen. o C) Step B) repeated for Days 2, 3, 4, 5 and 6.

• Temperature treatment o 0.25 ml of 20 mg/ml sample incubated at 4°C, 40°C or 50°C from Day 0. o 25 pl collected for analysis on Days 1 , 2, 3, 4, 5, and 6.

• Agitation treatment o 0.25 ml of 20 mg/ml sample agitated at 1000 rpm at room temperature from Day 0. o 25 pl collected for analysis on Days 1 , 2, 3, 4, 5, and 6.

• Oxidation treatment o 0.15 ml of 5 mg/ml sample treated with 0.1% or 0.01% H2O2 for 24 hours at room temperature and subsequently collected for analysis.

In further experiments, the effects of the antibody being provided at different concentrations was analysed for antibodies provided in buffer formulations F5 and F13:

• Concentration treatment o 0.25 ml of sample was provided at 20 mg/ml, concentrated to 50 mg/ml and 150 mg/ml and aliquots were subsequently collected for analysis.

5.3 Analysis

5.3.1 Protein quantification

Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop spectrophotometer, and a calculated extinction coefficient equal to 1 .4. For each sample, the respective formulation buffer was used to perform the blanking step.

The results obtained for antibody [3] are shown below. At 50°C temperature one sample presented some increased concentration at day 1 which could be due to evaporation (although all tubes were sealed with parafilm, some evaporation), or partial protein degradation or unfolding which may translate into changes in extinction coefficient. Overall, all other measured concentrations were within the range of the initial concentration (20 mg/ml).

Day 0:

Day 1 :

Day 3:

Day 6:

Oxidation treatments did not affect protein concentrations, as shown in the tables below.

No oxidation:

0.01% H2O2:

0.1% H2O2:

5.3.2 SE-HPLC

Size-exclusion high-performance liquid chromatography (SE-HPLC) analysis was performed using the UltiMate 3000 UHPLC system (Thermo Fisher). Samples were bracketed every 10 samples by one reference compound and one PBS blank injection. Mobile Phase: 0.2 M NaCI in 100 mM phosphate buffer pH 6.8; Column: MabPac-SEC 5 pm, 4 mm x 300 mm, Thermo. Flowrate: 0.23 mL/min.

Aggregation was determined by detection of a decrease in the relative percentage area of the main peak or an increase in relative % area of aggregates. Values were derived using auto determination mode, without any smoothing.

Results obtained for temperature, agitation and freeze/thaw treatments for antibody [1] formulations are shown in Figures 25A to 25E.

Results obtained for temperature, agitation and freeze/thaw treatments for antibody [3] formulations are shown in Figures 26A to 26E.

Results obtained for oxidation treatments for antibody [1] formulations are shown in Figure 27.

Results obtained for oxidation treatments for antibody [3] formulations are shown in Figure 28.

Results obtained for concentration treatments for antibody [1] formulations are shown in Figure 29.

Results obtained for concentration treatments for antibody [3] formulations are shown in Figure 30.

Most treatments did not significantly influence antibody integrity in the different formulations, with the exception of incubation at 50°C. Antibodies provided in formulation F13 were found to have the lowest propensity to aggregate when subjected to the different treatments.

5.3.3 CEX-HPLC

Cation exchange high-performance liquid chromatography (CEX-HPLC) analysis was performed to determine the distribution of charge variants of the antibodies utilizing Ultra-High-Pressure Liquid Chromatography method. Charge variants of the test article are separated by pH gradient chromatography using a cation exchange analytical column. Proteins with greater positive surface charge elute earlier than proteins with a less positive surface charge. Eluted charge variants are detected by UV 280 nm and the results for the main isoform, acidic peaks and basic peaks are expressed as the percentage of the total areas of the peaks.

10x CX-1 pH Gradient Buffer A (pH 5.6) (Thermo Scientific, #085346)

10x CX-1 pH Gradient Buffer B (pH 10.2) (Thermo Scientific, #085348).

Sample diluent: 10x CX-1 pH Gradient Buffer A (pH 5.6)

Column: YMC BioPro SP-F, 4.6 x 100 mm, 5 pm (YMC Co. Ltd., #SF00S05-1046WP) Antibody concentration in sample: 1 mg/ml Load: 35 pg

Flow rate: 0.8 ml/min

Results obtained for concentration treatments for antibody [3] formulations are shown below. Concentration of the antibody was not associated with changes to the charge distribution.

5.3.4 ELISA

384-well plates were coated overnight with 1 pg/ml of SARS-COV2 RBD protein from Sino Biological - Cat. 40592-V08H) in PBS at 4°C. Post incubation, plates were washed thrice with washing buffer (0.05% Tween 20 in 1x PBS ) and blocked for 1 h 30 min with blocking buffer (1% BSA in 1X PBS) at room temperature. Plates were subsequently washed once with washing buffer and incubated with an 11-point dilution series of the antibody (starting from 10 pg/ml, and using dilution factor of 5). After 1 h of incubation at room temperature, plates were washed thrice with washing buffer and incubated with a 1 :7000 dilution of HRP-conjugated secondary antibody (goat anti-Human IgG - HRP - Abeam, Cat. ab97225) for 1 h at room temperature, followed by three washes with washing buffer. Plates were developed with colorimetric detection substrate 3,3',5,5'-tetramethylbenzidine for 10 min. The reaction was stopped with ELISA Stop solution, and OD was measured at 450 nm using a BioTek PowerWave HT. An antibody known to bind to SARS-CoV-2 spike protein was included as a positive control, and lgG1 Isotype Lot: #UG2799151A, Invitrogen was used as a negative control.

Results obtained for temperature, agitation and freeze/thaw treatments for antibody [3] F5 and F13 formulations are shown in Figures 31 A to 31 C.

Results obtained for oxidation treatments for antibody [3] F5 and F13 formulations are shown in Figures 32A and 32B.

Results obtained for concentration treatments for antibody [3] F5 and F13 formulations are shown in Figure 33.

The different treatments were found not to have a significant impact on the ability of antibody [3] to bind to the RBD of SARS-CoV-2.

The inventors produced a cell line stably expressing antibody [3] of Example 1 .

6.1 Adaptation to culture in serum-free medium

CHO-k1 cells (ATCC, Cat. No. CCL-61) were first adapted to suspension culture in serum-free medium. Briefly, CHO-k1 cells were first cultured in F-12K medium supplemented with 10% heat- inactivated FBS (F-12K+10 medium). After two passages, the medium was exchanged to F75-25 medium (comprising 75% F-12K+10 medium, and 25% “50:50 medium”; “50:50 medium” is medium comprising 50% EX-CELL 325 PF CHO Serum-Free Medium + 50% EC-CELL CD CHO Serum-Free Medium + 6 mM L-Glutamine + 0.05% Pluronic F-68) with a seeding density of 5x10⁵ cell/mL. Cells were transferred to shake flasks and cultured in a 37°C, 5% CO2, humidified incubator with agitation at 110 rpm. After three passages in F75-25 medium, the cell culture was diluted into F50-50 medium (comprising 50% F-12K+10 medium, and 50% 50:50 medium) at seeding density 5x10⁵ cell/mL. The cells were passaged 6 times, and subsequently diluted to F25-F75 medium (comprising 25% F- 12K+10 medium, 75% 50:50 medium) at seeding density 5x10⁵ cell/mL. After two passages, the cell culture was diluted into 100% 50:50 medium with seeding density 5x10⁵ cell/mL for one passage, before two passages at a seeding density 2x10⁵ cell/mL. At the end of the process of adaption to culture in serum-free medium, the viability of cells in culture was 96.8%.

Cells were then cultured in EX-CELL Advanced CHO Fed-Batch medium (SAFC) supplemented with

6 mM L-glutamine (hereafter referred to as EX-CELL medium). Cells were diluted into EX-CELL medium at seeding density 2x10⁵ cell/mL, and cultured at 37°C in an 8% CO2 atmosphere, and 80% relative humidity incubator with agitation at 125 rpm.

6.2 Expression vector construction

A polycistronic expression vector encoding the heavy and light chains of antibody [3] of Example 1 was produced by cloning VH and VL region sequences codon-optimised for expression by CHO cells into MabDZ vector (described e.g. in US 2012/0301919 A1). A schematic representation of the polycistronic vector encoding antibody [3] is shown in Figure 34, and the sequence is shown in SEQ ID NO:101.

6.3 Transfection

Cells were transfected with the vector described in Example 6.2 above.

EX-CELL medium-adapted CHO-k1 cells were thawed and maintained at 37°C, 8% CO2, 80% relative humidity incubator, and 125 rpm agitation conditions for one week prior to transfection. 1x10⁷ cells were then seeded at a density of 5x10⁶ cell/mL, and electroporated with 5 pg of linearized expression vector using the 4D-Nucleofector kit (Lonza, Switzerland), electroporation program CA201 . Electroporated cells were incubated at 37°C, 5% CO2 humidified static cell incubator in 6-well plates containing 2 ml EX-CELL medium for 24 hr. Cells where then harvested by centrifugation and resuspended in selection medium comprising EX-CELL Advanced CHO Fed- Batch Medium + 6 mM L-Glutamine + 250 nM MTX + 200 pg/mL Zeocin, at a seeding density of 5x10⁵ cells/mL. Cells were transferred to fresh selection medium once per week. After four weeks, cells were transferred to maintenance medium comprising EX-CELL Advanced CHO Fed-Batch Medium + 6 mM L-Glutamine + 250 nM MTX.

6.4 Generation of MCB- 115-05

The MCB-115-05 stable clone was generated by limiting serial dilution from cells transfected and cultured as described in Example 6.3. MCB-115-05 was determined to produce large quantities of antibody [3], to grow rapidly and to have high viability in culture.

MCB-115-05 was deposited 5 November 2020 as ATCC patent deposit number PTA-126858.

General References

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m an

convalescent COVID-19

are essential for the

of the

Abstract

SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31 , a potent SARS-CoV-2 neutralizing IgG 1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18- human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFNy driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite FcyR binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development.

One Sentence Summary

Anti-SARS-CoV-2 IgG 1 antibody SC31 controls infection in vivo by blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.

Introduction

In December, 2019, a cluster of novel pneumonia (later named COVID-19) cases emerged and rapidly spread through human-to-human transmission in the city of Wuhan, China [1 , 2], High throughput sequencing of patient-derived samples revealed a novel beta-coronavirus, subsequently named SARS-CoV-2, as the etiological agent. SARS-CoV-2 was found to have 79.6% sequence homology to SARS-CoV, the virus responsible for an epidemic that caused 774 fatalities during 2002-2003 [3-6], Like SARS-CoV, SARS-CoV-2 has the potential to cause severe respiratory distress and significant mortality [1 , 2], While its natural reservoir remains unknown, based on sequence homology, SARS-CoV-2 is likely of bat origin [7], SARS-CoV-2 was found to bind to angiotensin converting enzyme 2 (ACE2), the same cellular surface receptor used by SARS-CoV, via the receptor binding domain (RBD) of the viral surface Spike protein (SP) [8], There is no preexisting immunity to SARS-CoV-2 due to its low homology to circulating endemic coronaviruses. This, coupled with its high human-to-human transmissibility, has led to an on-going global pandemic that has currently caused more than 40 million infections worldwide and over one million fatalities.

Antibodies derived from the memory B cells of recovered patients have become an attractive approach to developing therapeutic antibodies for infectious disease. Such antibodies have previously been found to be protective against coronavirus diseases, such as SARS and MERS, in animal models [9, 10], In particular, antibodies that blocked the viral SP protein from binding to ACE2 were highly potent at preventing infection [11 , 12], While such antibodies were not used clinically to treat coronavirus infections, antibodies derived from recovered patients had been successfully used to treat other infectious diseases, including the highly lethal Ebola virus disease with results superior to small molecule antivirals [13], This indicates the highly promising therapeutic potential of antibodies derived from convalescent patients, especially those that inhibit viral entry through the functional ACE2 receptor. The SARS-CoV-2 SP protein and its RBD have also become the focus of numerous accelerated vaccine development programs [14-16], However, given the challenges associated with large-scale roll-out of effective vaccines to the global population other options for protective immunity, even for shorter periods of time, must be considered. Therapeutic and prophylactic antibodies specific to SARS-CoV-2 have the potential to provide a viable treatment option before an effective vaccine is available, and further, can provide a much-needed treatment option for susceptible individuals who respond poorly to vaccination.

Multiple neutralizing antibodies against SARS-CoV-2 with therapeutic potential have been reported, with the most potent typically RBD-specific functioning by inhibition of binding to the ACE2 receptor [17-21], Several have been tested in murine, hamster and non-human primate (NHP) models, where they and demonstrated both prophylactic and therapeutic efficacy and a smaller number are currently progressing through clinical trials [22-24], In these pre-clinical models, therapeutic benefit was associated with reduced inflammation in the lung and correspondingly lower levels of proinflammatory cytokines and chemokines such as IL6, CCL2 and CXCL10, which have been observed to be elevated during the COVID-19 cytokine storm [25], Despite this, Antibody-Dependent Enhancement (ADE) of disease severity remains a major concern for the use of anti-viral antibodies as therapeutics. ADE can occur if Fey Receptor (FcyR) engagement mediates an increase in the infection of phagocytic cells that take up opsonized viral particles [26], Indeed, ADE has been observed in both in vitro and in vivo studies of SARS infection [27-29], Using mouse models, ADE has also been proposed to be a driver of the immune dysregulation observed in severe COVID-19 cases [26, 28], and therefore represents a risk for antibodies identified from a human immune response. Concerningly, in COVID-19 patients, higher anti-SP serum IgG levels have also been shown to correlate with hospitalization and severe disease [30, 31], however, there is no evidence to date that administration of convalescent plasma can lead to ADE.

As a result, several of the ongoing SARS-CoV-2 antibody programs have chosen to use Fc isotypes that do not engage FcyR, including the natural isotype lgG4 and engineered variants such as those carrying the LALA mutations [22], However, this may be counterproductive as the signaling mechanisms underpinning the efficacy of these antibodies have not been evaluated, particularly the potential for beneficial engagement of FcyR to induce a targeted anti-viral response.

We present here the discovery and development of a potent RBD-binding neutralizing lgG1 antibody, SC31 , originally isolated from an early convalescent patient. We have investigated the risk of ADE, and the impact of Fc functionality on its therapeutic efficacy via comparison with a FcyR null binding LALA variant and demonstrated that engagement of Fc receptors can drive a targeted antiviral response, does not cause ADE and is required for complete efficacy of SC31 in rodent models of severe disease. Isolation and development of a potent neutralisinq antibody, SC31, from an early convalescent

Anti-SARS-CoV-2 antibodies were generated by single B cell antibody interrogation. SP-binding lgG+ B cells were sorted directly from patient PBMC samples obtained at 15- and 27-days postsymptom onset and cultured to induce antibody secretion. Despite sampling soon after the onset of symptoms, SP-specific IgG B cells were detected at a frequency below 1% of total lgG+ B cells (Figure 41). Heavy and light chain antibody pairs were isolated from these clones and converted to full lgG1 antibodies.

Significantly, a comparison with IgG purified from the plasma fraction of the corresponding early convalescent patient sample showed that SC31 was 2000-fold more potent (Figure 35A). SC31 still showed complete and strong neutralization of Vero E6 infection when the quantity of SARS-CoV-2 used in the neutralization assay was increased 10-fold to 1 ,000 TCID50 (Figure 35B). SC31 bound to both the SP ectodomain and the RBD of SARS-CoV-2 with similar affinity (Figure 35C), indicating that inhibition of receptor binding is likely the mechanism of neutralisation. Indeed, SC31 demonstrated concentration-dependent inhibition of the interaction of both SARS-CoV-2 SP ectodomain and RBD with human ACE2 (Figure 35D). Taken together, SC31 is a potent neutralizing antibody that acts by inhibiting the interaction of SP RBD with ACE2.

To accelerate the translation from the discovery of SC31 as a potential therapeutic to a clinical development candidate, rapid development activities were conducted. Developability optimization was performed to remove known sequence liabilities, resulting in a lead sequence with significantly improved charge variant profile (Figure 44). Parallel establishment of high titer monoclonal master cell banks for multiple development variants of SC31 provided maximal optionality, pending functional and developability assessments, while producing sufficient material for in vivo functional studies, GLP toxicology studies, as well as process and analytical method development. A scale-up process for the lead MOB was rapidly developed that robustly yielded a gross titer above 4 g/L at the 2000L fed batch GMP production scale.

The epitope of SC31 is located at the ACE2 interface of the SARS-CoV-2 SP protein and conserved in all circulating SARS-CoV-2 mutants

To investigate the structure and function of the epitope recognized by SC31 , the binding of SC31 to naturally occurring SP mutants was evaluated. A total of 36 single amino acid SP mutations identified from publicly available databases were tested [32, 33], We focused on 23 mutations that were within the receptor binding motif (RBM, aa438-506) and predicted to make direct contact with ACE2 receptor, or that were high frequency mutations beyond the RBM. The majority of these mutations were found to have minimal effect on SC31 affinity to SP and its binding to the ACE2 receptor (Figure 42). Significantly, the two most common circulating mutations within the RBM, N439K and S477N, as well as the D614G mutation which delineates a major viral clade and is by far the most frequent SP mutation did not cause any significant changes in affinity (Figure 36A). Six mutations (I434K, S438F, D467V, L455F, A475V, N501Y) were identified that resulted in significant loss of SC31 binding. Three of these six mutations, I434K, S438F and D467V, also resulted in significant or total loss of binding to the ACE2 receptor (Figure 36B). This suggests that the loss of binding was due to destabilisation of the entire RBM structure and that these mutants would be unlikely to propagate further due to reduced fitness. The other three mutations minimally reduced or even enhanced ACE2 receptor binding, as in the case of N501 Y, and could be expected to lead to viral escape due to an alteration of the binding epitope. The locations of these three mutations are highlighted on a recently published crystal structure [34] and are likely to represent part of the SC31 binding epitope (Figure 36C). Critically, none of these 3 SP mutants which appeared several months prior appear to be commonly circulating, perhaps due to other negative effects on the stability of the protein. SC31 therefore binds to a stable epitope in the RBD of the SARS-CoV-2 SP protein that is well conserved in all commonly circulating SARS-CoV-2 variants.

SC31 requires Fc-mediated effector mechanisms for maximal therapeutic efficacy in the SARS-CoV- 2 K18-hACE2 mouse severe disease infection model but does not cause ADE

SC31 is an lgG1 antibody therefore binding of the Fc domain to FcyR on immune cells is expected. FcyR binding may stimulate beneficial Fc-mediated effector function, including Antibody-dependent Cellular Cytoxicity (ADCC), Antibody-dependent Cell Phagocytosis (ADCP), Antibody-dependent cell-mediated virus inhibition (ADCVI) and Complement-Dependent Cytoxicity (CDC), but, as previously discussed, may also lead to ADE for viral infection. ADE is especially important when antibodies reach sub-neutralising concentrations. To ameliorate the risk of ADE for anti-viral antibodies, it is possible to utilize an Fc isotype that does not bind to FcyRs, however, this could also impact the potency of the antibody if Fc-mediated effector functions contribute to the therapeutic efficacy of the antibody.

To first establish if Fc-mediated effector function could have a role in the therapeutic efficacy of SC31 , the ability to induce Fc effector-mediated activity was investigated by comparing against an FcyR null-binding variant (LALA) of the antibody. The upstream activation of the FcyRllla ADCC signalling pathway was evaluated using a Jurkat reporter cell line co-cultured with target HEK293 cells expressing membrane-bound SARS-CoV-2 SP. Here, SC31 was confirmed to induce dosedependent activation of ADCC signalling. In contrast, there was no activation of ADCC observed with the FcyR null-binding variant (LALA) of the antibody, similar to the mock-transfected HEK293 cells (without SP) (Figures 37A and 37B) [35],

Subsequently, the therapeutic efficacy of SC31 and its LALA variant were evaluated in a SARS- CoV-2 K18-hACE2 mouse model to investigate if Fc effector mechanisms contributed to therapeutic efficacy. Severe disease manifestations with SARS-CoV-2 infection have been demonstrated in K18-hACE2 transgenic mice [21 , 36-38], Mice infected intranasally with 1.2 x 104 TCID50 (nCoV- 19/Singapore/3/2020) presented with severe disease including lethargy, weight loss, overexpression of proinflammatory cytokines/chemokines (IL-6, CXCL10, and CCL2) and, ultimately, death between

6- and 8-days post infection (dpi) with associated high virus titers in lung tissue. (Figure 43).

SC31 and the LALA variant (20mg/kg) were introduced by intraperitoneal (IP.) administration 6 hours post virus infection (hpi). At 3 dpi, lungs from half of the mice per group were harvested for total and infectious viral load quantification and cytokine/chemokine mRNA and protein expression analysis. The remaining mice were monitored for survival and weight for a further 15 days (Figure 37C). Quantification of viral RNA and infectious virus showed that the LALA variant was somewhat less efficacious than the wild-type lgG1 in controlling viral load (Figure 37D). However, in contrast, significantly more severe illness was observed in the virus-infected mice treated with the LALA variant, demonstrated by their greater weight loss and higher mortality (Figure 37E). Observations of the pro-inflammatory cytokine IL6 and chemokines CXCL10 and CCL2 from virus-infected mice showed that, while there was a similar reduction in IL6 for both IgG 1 and the LALA variant, the LALA mice showed significantly higher levels of CXCL10 and CCL2 to those treated with lgG1 (Figure 37F). Where both data were available the protein levels mirrored the mRNA levels. Intriguingly the protein levels of IFNy were markedly increased in the lgG1 group, suggesting that although the systemic pro-inflammatory response is decreased, there is likely a beneficial targeted anti-viral response. Taken together these results indicate that in addition to the ability to inhibit the binding of SARS-CoV-2 to ACE2, Fc-mediated modulation of the immune response is critical for optimal therapeutic benefit.

Finally, to investigate the risk of ADE, SC31 and its LALA variant were tested at sub-neutralizing concentrations using a SARS-CoV-2 pseudovirus and FcyRllla-expressing THP-1 and Raji cell lines. ADE has been previously been modelled for SARS-CoV pseudovirus in these same cell lines [28, 39], Importantly, no pseudovirus infection was observed in both THP-1 and Raji cells for either antibody at all concentrations tested, indicating that SC31 , despite its FcyR binding leading to potent Fc mediated effector functions, is unlikely to mediate ADE (Figures 38A and 38B). It has been suggested that pH selective binding of anti-viral antibodies, i.e. lower binding affinity to the viral target at lower pHs may predict a risk of ADE as antibodies could dissociate from the virus in the low pH environment of the endosome and release the pH resistant virus to enter the cell. We found that SC31 maintains a high affinity for SP down to pH 4.5 and this may be related to the lack of observable ADE for this antibody (Figure 38C).

SC31 provides potent dose dependent therapeutic benefit in the SARS-CoV-2 K18-hACE2 mouse severe disease infection model

Understanding the minimum therapeutic dosing level and optimal timing of an anti-SARS-CoV-2 antibody therapy for the treatment of COVID-19 remains critical to evaluate the potential of such an antibody in a clinical setting. To further evaluate the therapeutic potency and dose response of SC31 , mice were treated with 2, 5, 10, or 20mg/kg doses 6 hpi. At 3 dpi, half the mice were sacrificed for quantification of lung viral RNA, infectious virus, and cytokine/chemokine expression. The remaining mice were monitored for survival and weight for a further 25 days (Figure 39A). A dose-dependent reduction in weight loss, starting at 3dpi, was observed in all virus infected mice. A similar dose-dependent effect on survival was observed, with no mortality in animals treated with the highest dose (20mg/kg), 50% mortality in mice dosed with either 5 or lOmg/kg and no survival at the lowest dose (2mg/kg). All surviving mice overcame clinical signs of disease by day 13, as evidenced by the return to their pre-infection weight (Figure 39B). Quantification of SARS-CoV-2 RNA in lung tissue showed a dose-dependent reduction of viral RNA ranging from 0.8 to 1 .5-log from the lowest to highest dose, respectively (Figure 39C). A corresponding reduction in infectious virus was observed with a greater than three log reduction with 5mg/kg antibody and down to the limit of detection for the majority of mice dosed at 20mg/kg (Figure 39D). In the 10 and 20mg/kg treatment groups, pro-inflammatory cytokine IL6 and chemokines CXCL10 and CCL2 showed a decrease to levels similar to that in uninfected mice (Figure 37E). Taken together, these results suggest that SC31 has therapeutic benefit at doses above 5mg/kg.

To ascertain the efficacious dosing window for SC31 , mice were treated with 20mg/kg of antibody at 6, 24 and 48 hpi with half the mice sacrificed for lung viral load and cytokines at 3 dpi and the remainder monitored until Day 15 (Figure 40A). All mice treated at 6 and 24 hpi survived with minimal weight loss while mice treated at 48 hpi lost weight and succumbed to disease at the same rate as untreated mice (Figure 40B). Lung viral RNA, infectious virus and IL6, CXCL10 and CCL2 levels followed the same trend with similar therapeutic benefit observed in mice treated at 6 and 24 hpi while mice treated at 48 hpi exhibited values similar to untreated mice (Figures 40C to 40E). Taken together, the results indicate that the efficacious dosing window in this model is before 48 hpi, i.e., prior to the peak of viral infection and inflammation that was also observed at day 3 (Figure 43).

Discussion

There is an urgent need for development of safe and efficacious therapeutic antibodies to address the global COVID-19 pandemic. Such antibodies represent a potentially critical treatment option in the absence of a widely available vaccine especially for elderly and other at-risk groups. Nevertheless, relatively few antibody candidates have progressed to clinical evaluation to date and only a few have reported potency data in pre-clinical models [18, 21 , 24, 40], This is, in part due to the challenges of demonstrating that any clinical candidate is both safe and potent enough to predict clinical benefit, which requires the rapid development and scale-up manufacture of a stable drug-like antibody, and evaluation in complex animal models. Further, concern over the possibility of ADE has led some efforts to focus on potentially sub-optimal Fc-effector silent candidates (lgG4 or antibodies with Fc mutations that abolish FcyR binding). Although data is still emerging, two lgG1 antibody programs [41 , 42] are already reporting promising early data in clinical trials, but there is limited information on their mechanism of action and the possibility of ADE at sub-therapeutic doses is still an open question. In this study we present the generation and characterization of a highly efficacious anti-SARS-CoV-2 lgG1 neutralizing antibody, SC31 . The binding site of SC31 has been mapped to the receptor binding motif of the SP protein and has been shown to be conserved across all common circulating SARS-CoV-2 mutants, including the most common variant, D614G. In parallel to the rapid development and scale-up manufacture of the SC31 antibody, the role of Fc-mediated effector activity on the therapeutic efficacy of the antibody was evaluated alongside the potential to cause ADE in order to establish the optimal antibody format for evaluation in the clinical setting.

Although lgG1 isotype antibodies are predicted to bind stimulatory FcDR and trigger signaling as well as Fc-mediated effector functions, such as ADCC, ADCVI and CDC, not all lgG1 anti-SP antibodies exhibited the same capacity to elicit Fc-mediated effector functions. Specifically, it has been shown that some anti-SP IgG 1 antibodies exhibited weak or even negligible ADCC activity [17, 43], This may explain the variation between in vitro and in vivo potency reported for different anti-SP antibodies as well as a potential reason for the lack of immunity afforded by higher anti-SP-IgG titres in some COVID-19 patients [30], This also suggests that the ability of an antibody to promote Fc- mediated effector functions and signaling may be another key factor in therapeutic efficacy in addition to neutralization potency. Importantly, the SC31 antibody was clearly able to trigger Fc- mediated effector functions, as evidenced by activation of ADCC signaling in contrast to a Fc- effector null LALA variant, but notably, despite this, SC31 showed no evidence of ADE at sub- therapeutic doses, identical to the LALA variant.

This ability of SC31 to induce Fc-mediated effector signaling was subsequently demonstrated to be essential for the optimal therapeutic efficacy. When SC31 and its LALA variant were evaluated in an in vivo mouse model of SARS-CoV-2 infection, the LALA variant was observed to have much reduced therapeutic efficacy with only 50% survival compared with 100% survival for SC31 . This correlated with higher lung viral load as determined by both viral RNA and infectious virus, suggesting that the LALA variant was not able to clear virus, thereby triggering higher inflammatory responses. This indicates that FcyR engagement of lung phagocytic cells by SC31 not only did not result in excessive pro-inflammatory signaling, but appeared to induce a more targeted and robust anti-viral response characterized by higher IFNy levels. The reduction in systemic pro-inflammatory markers including IL6, CCL2 and CXCL10 is likely due to the reduction in viral load that dampens the systemic inflammatory signaling by various innate viral pathogen recognition receptors such as Toll-Like Receptor ? [44], The increase in IFNy levels, however, may reflect the engagement of a targeted NK and T-cell driven anti-viral response, as these are the primary producers of IFNy. In summary, although a recent study showed that anti-SP IgG from COVID-19 patients with severe disease can promote hyperinflammatory responses in alveolar macrophages [45], our data indicates that SC31 is unlikely to pose a risk of systemic immune dysregulation when used as a therapeutic.

Severe disease and mortality in COVID-19 appears to be driven by excessive inflammation due to failure of the immune system to control viral infection in the lung [46], SC31 showed potent dosedependent therapeutic efficacy above 5mg/kg, however, once the inflammatory cascade is triggered, SC31 was no longer able to exert a therapeutic effect as evidenced by the poor outcome when mice were treated 48 hpi. This indicates that SC31 antibody therapy is best administered prior to the onset of severe symptoms.

Overall, we have demonstrated that the anti-SP IgG 1 antibody SC31 , generated from an early convalescent patient at day 27 after symptom onset, is able to control infection in two animal models of COVID-19 disease by decreasing viral load and protecting against lung damage, and has the potential to be a highly efficacious therapeutic in the clinical setting. This efficacy is driven by the dual mechanisms of potent neutralization of SARS-CoV-2 infection through blocking SP binding to the human ACE2 receptor, and induction of a robust anti-viral response driven by Fc-mediated effector functions, but importantly, without concomitant ADE. Furthermore, we have shown that SC31 is efficacious against two circulating strains of SARS-CoV-2. Following a highly accelerated non-clinical and CMC development program that has resulted in an efficient scaled-up manufacturing process yielding over 4g/L in large scale GMP manufacture, SC31 , also known as HMBD-115 and AOD01 for development, will shortly begin human trials in COVID-19 patients.

Materials and Methods to Example 7

Single cell sorting and culture

Ficoll-Paque patient PBMCs at 5x10⁶cells/ml concentration were incubated with 10ug/ml Twin-strep- tagged WT-spike for 1 hr at 4°C in FACS buffer (1x PBS, 5mM EDTA, 1% fetal calf serum), washed in 1xPBS and then stained with fluorescently labelled antibodies at the following concentrations (5pl anti-HuCD19-Pacific Blue, 5pl anti-HuCD27-Alexa647, 2.5pl-anti-HulgG-BV711 , 5pl anti-HuCD38- PE-Cy7, 2.5pl anti-Strep-tagll-Alexa488 in 10OpI of FACS buffer per 10⁶ PBMC cells) for 30min at 4°C. Cells were washed with 1xPBS and resuspended in FACS buffer containing 1 :100 propidium iodide (PI) before sorting on a FACSAria Fusion. PF, CD19⁺, lgG⁺’ Spike⁺ cells were sorted individually into 96-well plates containing 50pl of IMDM culture medium, 5x10³ 3T3-msCD40L feeder cells [Huang, J., et al., Isolation of human monoclonal antibodies from peripheral blood B cells. Nat Protoc, 2013. 8(10): p. 1907-15] and supplements and cytokines at concentrations previously described without CpG and addition of IL-21 at 10ng/ml [Jourdan, M., et al., Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation. J Immunol, 2011. 187(8): p. 3931-41], Cells were spun down after 8 days of culture and stored with 10pl of lysis buffer (QuickExtract RNA Extraction Solution, Lucigen) and supernatants tested by ELISA and microneutralization.

Antibody and viral protein expression and purification

Antibody variable heavy and light chain sequences were cloned into a pCMV-promoter driven expression plasmid containing the appropriate human lgG1 heavy, kappa or lambda light chain constant regions as well as a leader sequence for secretory expression. For spike protein expression, the complete ectodomain including the leader sequence (Accession No. MN908947, S gene amino acids 1 -1208) together with a C-terminal Twin-strep tag (WSHPQFEK- GGGSGGGSGGS-SAWSHPQFEK), replaced the antibody sequence. Point mutations to spike protein were subsequently introduced using Quikchange site-directed mutagenesis kit (Agilent). For RBD expression, only amino acids 331-524 were cloned in together with Twin-strep tag and the leader sequence. Purified plasmid was transfected into HEK293 suspension cell culture in F17 media (ThemoFisher) at a concentration of 1 .Omg DNA/L of culture with branched polyethylenimine (Sigma-Aldrich) at 3:1 w/w ratio of PEI:DNA. Culture supernatant was harvested after 7 days and purified by HPLC using StrepTactinXT Superflow High Capacity Column (IBA Life GmBH) or MabSelect SuRe Column (Cytiva) for viral proteins or antibodies respectively.

Virus culture and microneutralization

SARS-CoV-2 virus obtained from a patient nasal swab was cultured in VeroE6 cells and supernatant harvested on observation of 90% CPE (hCoV-19/Singapore/3/2020). Antibodies at indicated concentrations was incubated with 100 TCIDso of virus and 2x10⁴ VeroE6 cells in 10OpI of culture media (MEM/2% FCS) in 96-well flat bottom plates and incubated for 72hrs. Neutralization was measured using Viral Toxglo reagent (Promega) to determine percentage cell survival relative to a no virus and virus only controls. For initial screening of B cell supernatant, 12.5pl of supernatant was mixed with 25 TCIDso of virus instead.

ELISA

To determine antibody affinity to RBD, WT or mutant spike protein, 2ug/ml purified protein in binding buffer (100 mM Tris-HCI, 1 mM EDTA, 150 mM NaCI, pH8.0) was coated onto Streptactin XT 96- well ELISA plates (IBA GmBH) for 2hrs. Plates were washed with PBS and antibody diluted into 2% BSA/PBS blocking solution at indicated concentrations then incubated for 1 hr before washing thrice with PBS/0.05%Tween. For binding at different pH, antibodies were incubated in 1xPBS adjusted to the appropriate pH with HCI with 0.5% BSA as block. Antibody binding was detected using 1 :5000 anti-huIgG Fc-HRP conjugated secondary antibody (ThermoFisher) diluted in blocking solution incubated for 1 hr. Plates were then washed thrice with PBS/0.05% Tween and once with PBS. After washing, plates were developed with colorimetric detection substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce). The reaction was stopped with 2M H2SO4, and OD was measured at 450 nm

Binding inhibition assay

20mM Twin-strep-tagged viral protein was incubated with antibody for 1 hr in 50pl FACS buffer. 5x10⁴ huACE2 expressing CHO cells (a kind gift from A/Prof Dr Tan Yee Joo, National University of Singapore) in 50ul FACS buffer was added and incubated for a further 1 hr. Cells were spun down at 300g for 5min, washed twice with PBS and then stained with 1 :50 Alexa488 conjugated anti-Strep- Tag II antibody (StrepMAb-lmmuno, IBA GmBH) for 30min in 50p I FACS buffer. All incubations were carried out at 4°C. Cells were finally washed once with 1x PBS and stained with 1 :100 propidium iodine (PI) and analysed on a BD FACS Canto II. PI- cells were gated and binding measured by 488 channel fluorescence intensity. Charge Variant Analysis

Briefly, for charge variants, 35 ug protein samples were loaded onto a HPLC system (Thermo Fisher) coupled with an analytical cation exchange column (YMC BioPro SP-F, 4.6 x 100 mm, 5 urn) and UV detector (VWD) at UV length 280 nm under 0.8 mL/min constant flowrate. The proteins samples are eluted by pH gradient (25% - 45% Mobile phase B over 22 mins, Mobile phase A: CX-1 pH 5.6, Mobile phase B: CX-1 pH 10.2, Thermo Fisher), with the acidic surface charged protein elutes earlier than basic surface charged molecule. The relative abundance of acidic variants, basic variants and main isoform are reported. Sample are injected in duplicates.

Isoelectric point determination

Isoelectric point was determine using the PA800 Plus system coupled with UV detector (Beckman Coulter), the protein samples were desalted by buffer exchange to 20 mM Tris pH 8.0 using protein concentrator (Amicon, Merck) and mixed with a mixture of 40 mM arginine, 1 .6 mM iminodiacetic, 2.4 M urea, and 4.8% pharmalyte 3-10, as well as 0.8% pl marker 10.0, 9.5 and 4.1 , at 0.2 mg/mL final concentration. The mixture was then injected into a neutral-coated capillary with one end submerged in anolyte (phosphoric acid) and the other submerged with catholyte (sodium hydroxide). The molecule migrates to its isoelectric point during the focusing step (15 mins, 25 kV) and was followed by a 30 mins mobilisation phase at a voltage of 30 kV. The A280 signals were collected to determine the main peak pl.

Antibody Dependent Cellular Cytotoxicity (ADCC)

Antibody Dependent Cellular Cytotoxicity was tested using a Jurkat reporter cell line stably expressing FcyRHIa and an NFAT response element driving downstream expression of firefly luciferase as per manufacturer’s protocol (ADCC reporter assay, Promega). Target cells were generated by transiently transfecting HEK293 suspension culture with the full-length WT-spike construct including the transmembrane domain but lacking the C-terminal 19 amino acids which contains an endoplasmic reticulum (ER)-retention signal that had been found to reduce incorporation into pseudovirus [Fukushi, S., et al., Vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein. J Gen Virol, 2005. 86(Pt 8): p. 2269-2274], Cells were harvested after 72hrs and seeded at 25,000 cells/well and at 1 :3 ratio with reporter cells and purified IgG incubated at indicated concentrations. Luminescence was measured after 6hrs incubation.

Antibody Dependent Enhancement

Spike-bearing viral pseudoparticles were produced through co-transfection with the above full length WT-spike construct along with the lentiviral plasmids pMDLg/pRRE, pRSV-REV (a kind gift from Dr Wang-Cheng-I, Singapore Immunology Network) and the luciferase reporter plasmid pHIV-Luc [Dull, T., et al., A third-generation lentivirus vector with a conditional packaging system. J Virol, 1998. 72(11): p. 8463-71] into HEK293 adherent cells and harvested after 4 days. 5pl of pseudovirusbearing supernatant was mixed with antibody at indicated concentrations and Raji, THP-1 or ACE2 expressing CHO-cells at 25,000cells/well and incubated at 37°C in a CO2 incubator. Media was changed after 24hrs and luminescence expression measured after a further 24hrs by washing the cells in PBS and adding reagent (Luciferase Assay System, Promega)

Efficacy testing for SARS-CoV-2 infection in K18-ACE2 mice

All animal work was monitored by and performed in accordance with the protocol approved by the DSO Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). B6.Cg-Tg (K18-ACE2)2Prlmn/J) were obtained from Jackson Laboratory (JAX). Mice for the studies were female between 7 and 12 weeks old. To determine the day of peak viral load, following acclimatization of the mice to the isocages, groups of 3 mice were anesthetized individually with 3% isoflurane using the precision vaporizer and infected intranasally (LN.) with 50pl of 1 .2x10⁴ TCID50 of SARS-CoV-2. Following infection, the mice were transferred to new isocages. On the indicated days three mice were euthanized by carbon dioxide asphyxiation, the lungs harvested, weighed and made to 10%w/v with viral grow medium then mashed through a disposable mesh using a plunger and aliquoted into screw cap tube and stored at -80° C, for later determination of lung viral load by qualitative real-time PCR (qRT-PCR) and cell culture to determine the tissue culture infective dose (TCID), and cytokine/chemokine mRNA expression. To assess therapeutic efficacy, at the indicated time-point mice were anesthetized with 3% isoflurane using a precision vaporizer and treated with indicated concentration of antibody in 200ul PBS by intra-peritoneal (I.P.) injection, and mice were returned to the isocages for recovery. Lungs were harvested for viral load and cytokine/chemokine mRNA expression at the peak virus day. For the survival groups mice were weighed when indicated and returned to their isocage. Any mice that showed >20% weight loss or significant inactivity were euthanized humanly using carbon dioxide asphyxiation.

Determination of lung viral load in infected mice

Lung viral load was determined using Tissue Culture Infection Dose (TCID50) in VERO E6 cells, or by real-time Polymerase Chain reaction (RT-PCR) detecting viral RNA (genome copy number;

GCN). Briefly for TCID50, serially-diluted lung homogenates were incubated with 2x10⁴ Vero E6 cells in total of 10Oul of culture media (MEM/2% FCS) in 96-well flat bottom plates and incubated for 5 days. Virus titre, reciprocal to cell viability, was measured using Viral Toxglo reagent (Promega) to determine cell viability relative to uninfected (cells only) controls. TCID50 was subsequently determined using Reed-Muench method. To determine the viral GCN, RNA was extracted from lung homogenates using QIAamp Viral RNA mini kit (Qiagen). Detection of viral RNA was achieved using primers and probes targeted against ORFI ab as described in [Chia, P.Y., et al., Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients. Nat Commun, 2020. 11 (1): p. 2800] with 7500 Fast Real-Time PCR system (Applied Biosystem). GCN was determined against standard controls included within each RT-PCR run.

Cytokine and chemokine mRNA measurements RNA was extracted from lung homogenates harvested at 3 dpi using QIAamp Viral RNA mini kit. cDNA was synthesized using High-Capacity cDNA reverse transcription kit (ThermoFisher) with addition of RNase inhibitor (RNaseOUT, ThermoFisher). Cytokine and chemokine expression was determined using TaqMan Fast Universal PCR mastermix (ThermoFisher) with PrimeTime® Standard qPCR assays (Integrated DNA Technologies) for CCL2 (Mm.PT.58.42151692), CXCL10 (Mm. PT.58.43575827), IL1 b (Mm.PT.58.41616450), IL6 (Mm.PT.58.10005566), TNF

(Mm. PT.58.12575861), IFNyl (Mm.PT.58.30132453.g) and normalised to GADPH (Mm.PT.39a.1) levels. Fold change was determined using the 2 ^DDCt method comparing anti-SARS-CoV2 specific or isotype control monoclonal antibodies-treated I irrelevant isotype treated mice, to uninfected mice controls.

Cytokine and chemokine protein measurements

Cytokine and chemokine protein levels in mouse serum were determined by ELISA using paired antibodies for mouse IFNy (Invitrogen, Cat# 88-7314), IL2 (Invitrogen, Cat# 88-7024), IL6 (Invitrogen, Cat# 88-7064) and CCL2 (R&D Systems, Cat# DY479-05), according to the manufacturer’s instructions. Briefly, 384 well plates were coated with 1X capture antibody for 16hr at 4°C. After blocking for 1 hr with blocking buffer provided in the kits, mouse serum was added to the plate and incubated for 2hr at room temperature. Plates were washed thrice with washing buffer and incubated for 1 hr with detection antibody, followed by 1 hr incubation with Streptavidin-HRP. After washing, plates were developed with colorimetric detection substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce). The reaction was stopped with 2M H2SO4, and OD was measured at 450 nm.

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Separately, Chan CEZ et al., PLoS One. 2021 ; 16(6):e0253487, which is hereby incorporated by reference in its entirety, describes that SC31 has been found to provide potent therapeutic benefit in Golden Syrian hamsters infected with SARS-CoV-2, protecting against severe COVID-19-like disease, and to provide therapeutic benefit and eliminate infectious virus in a rhesus macaque model of COVID-19.

The primary objective was to assess the safety and tolerability of ascending doses of intravenously (IV) administered SC31 in healthy adult volunteers.

The secondary objective was to assess the pharmacokinetic (PK) profile of IV administered SC31 in healthy volunteers and to assess the immunogenicity of IV administered SC31 in healthy volunteers.

Primary: The incidence and severity of treatment emergent adverse events (TEAEs) in subjects treated with SC31 or placebo, reported between the time of first dose of study drug and the end-of-study visit [Time Frame: Day 1 through Day 92]

Secondary: Pharmacokinetic parameters including, but not limited to, Cmax, AUCO-t, AUCO-inf,

Tmax, t1/2, etc. [Time Frame: Day 1 through Day 92], Anti-drug antibody to SC31 [Time Frame: Day 1 through Day 92]

Tertiary: Ex-vivo bio-assay for clinical samples. Immunomonitoring of clinical samples for changes in cytokines, immune cells and RNA tempus.

The study was a prospective, blinded, randomised, placebo-controlled, ascending dose study in healthy adults.

Healthy volunteers were screened for protocol specified eligibility criteria, and eligible subjects were randomised in sequential ascending dose cohorts, including four single dose cohorts and one multiple dose cohort. Each subject recruited to the study had a screening period from Days -28 to -1 , a single dose administered by infusion over 60 minutes (on Day 1 in single dose cohorts and on Day 1 and Day 2 in the multiple dose cohort), an inpatient observation period of 5 days after the last infusion and a follow-up period from the day of discharge to end of study (EOS) on Day 92. The safety observation period in each cohort was 72 hours after the last infusion. A sentinel group of at least two subjects was evaluated for the first cohort, fourth and the fifth cohort, before the remainder of subjects in these cohorts will be dosed.

Study cohorts and dose escalation

The calculated maximum recommended starting dose (MRSD) is based on the minimum anticipated biological effect level (MABEL) estimated from animal efficacy studies. The SARS-CoV-2 viral infection challenge in k18-hACE2 mouse model indicated that the pharmacologically active dose was equal to or more than 5 mg/kg, and a dose lower than 5 mg/kg would have minimal activity. A dose of 2 mg/kg has thus been selected as the starting clinical dose.

The study investigated 5 cohorts: 4 single dose cohorts at dose levels of SC31 (Dose 1 : 2 mg/kg, Dose 2: 5 mg/kg, Dose 3: 10 mg/kg, and Dose 4: 20 mg/kg), and one multiple dose cohort (at the highest safe dose studied in the single ascending dose cohort), see Table below. A total of 5 healthy volunteer groups were enrolled.

Table 1 : Dose levels and cohorts

at the highest safe dose studied in the single ascending dose cohort.

Additional cohorts of similar composition to Cohort A2 may be added to the study in order to address any safety questions that may arise. Intermediate doses can be selected based on emerging safety data.

Cohorts will overlap. Each new dosing level can begin after 72 hours has passed from the start of dosing and at least 48 hours of safety, tolerability, and preliminary PK data (if available) from each subject in the prior dose cohort, has been assessed by the safety committee, and deem that there have been no significant adverse events in the previous dosing level that would preclude dosing at a higher dose. Similarly, the multiple dose cohort begins after 72 hours has passed from the start of dosing and at least 48 hours of safety, tolerability, and preliminary PK data (if available) from each subject at the highest dosing level in single dose cohorts, has been assessed by the safety committee, and a highest safe dose has been determined. Subjects will be given a placebo or SC31 at the highest safe dose determined in single dose cohorts. There will be a sentinel group of two subjects.

Sentinel groups have been denoted for some of the cohorts. This group is indicated by the cohort number followed by a lowercase ‘s’. Additional sentinel groups of at least 2 subjects (1 active, 1 placebo) may be added to any cohort in any part if there is clinical need or increased safety concerns upon recommendation by the safety committee. If a cohort includes a sentinel group, the remainder of the cohort may be dosed only after at least 24 hours has passed since the start of dosing of the last subject in the sentinel group and there is at least 12 hours of safety data and at least 24 hours of observation data with no significant abnormalities that would raise concern about dosing other healthy adult volunteers. Alternatively, dosing may be staggered across the cohort by breaking the cohort into dosing groups of no more than two subjects each. If staggered dosing is used, the first group must have one placebo, similar to the composition of a sentinel group. Each group may begin only after at least 24 hours has passed since the start of dosing of the last subject in the previous group and there is at least 12 hours of safety data and at least 24 hours of observation data with no significant abnormalities that would raise concern about dosing other healthy adult volunteers.

Alternatively, dosing may be staggered across the cohort by breaking the cohort into dosing groups of no more than two subjects each. This may also be done to facilitate study operations at the site(s). This will be agreed upon in advance by the safety committee and will be influenced by new safety data. If staggered dosing is used, the first group must have one placebo, similar to the composition of a sentinel group. Each group may begin only after at least 24 hours has passed since the start of dosing of the last subject in the previous group and there is at least 12 hours of safety data and at least 24 hours of observation data with no significant abnormalities that would raise concern about dosing other healthy adult volunteers.

Approximately 40 participants were screened to achieve 23 randomly assigned to study treatment.

Treatment groups and duration

The screening period was up to 28 days and study duration of up to Day 92, for a total of up to 119 days for each subject. Subjects received study drug or placebo. The overall study duration for the study was up to 20-24 weeks.

Key inclusion and exclusion criteria

Key inclusion criteria: • Participant must be 21 to 55 years of age inclusive, at the time of signing the informed consent

• Male or female participants who are overtly healthy as determined by medical evaluation including medical history, physical examination, laboratory tests, and cardiac monitoring

• Body weight within 50-100 kg and body mass index (BMI) within the range 18 0-35 0 kg/m² (inclusive)

Key exclusion criteria:

• Significant allergies to humanised monoclonal antibodies

• Has laboratory-confirmed SARS-CoV-2 infection, or was previously infected with SARS- CoV-2, as determined by polymerase chain reaction (PCR), serological assays or other commercial or public health assay in any specimen prior to randomisation

• Evidence of active or latent tuberculosis (TB) as documented by medical history, examination, and either chest X-rays (posterior anterior and lateral), and/ or TB testing

• Alanine transaminase (ALT) >1 ,5x upper limit of normal (ULN)

• Bilirubin >1 .5xllLN (isolated bilirubin >1 .5xllLN is acceptable if bilirubin is fractionated and direct bilirubin <35%)

• QTc >450 ms for male participants or >470 ms for female participant

• Past or intended use of over-the-counter or prescription medication [including herbal medications] within 7 days prior to dosing

• Have hepatitis B, C or HIV infections

• Regular use of known drugs of abuse

• Regular use of alcohol (average weekly intake of >28 units for males or >14 units for females)

• History or presence of cardiovascular, respiratory, hepatic, renal, gastrointestinal, endocrinological, haematological, or neurological disorders capable of significantly altering the absorption, metabolism, or elimination of drugs; constituting a risk when taking the study intervention; or interfering with the interpretation of data

• Clinically significant multiple or severe drug allergies, intolerance to topical corticosteroids, or severe post-treatment hypersensitivity reactions (including, but not limited to, erythema multiforme major, linear immunoglobulin A [IgA] dermatosis, toxic epidermal necrolysis, and exfoliative dermatitis)

• Lymphoma, leukaemia, or any malignancy within the past 5 years except for basal cell or squamous epithelial carcinomas of the skin that have been resected with no evidence of metastatic disease for 3 years

• Current or chronic history of liver disease or known hepatic or biliary abnormalities (with the exception of Gilbert's syndrome or asymptomatic gallstones)

• Abnormal blood pressure as determined to be clinical significant by the investigator • Live vaccine(s) within 1 month prior to screening, or plans to receive such vaccines during the study

• Treatment with biologic agents (such as monoclonal antibodies including marketed drugs) within 3 months or 5 half-lives (whichever is longer) prior to dosing

Treatments administered

Table 2: Treatments administered

*ln case of infusion reactions the duration can be extended up to 4 hours. For the multiple dose cohort there will be a second dose on Day 2 (24 hrs after last infusion).

Pharmacokinetic analyses

Pharmacokinetic parameters are calculated for each subject with sufficient PK data for analysis using standard non-compartmental methods. The parameters AUC(O-tlast), AUC(0-«), AUC(O-tau), t1/2, CL/F, V/F, Tmax, and Cmax are calculated from plasma SC31 concentration-time profiles.

Figure 45 shows mean (SE) serum concentrations of SC31 versus time at the four dose levels. Data suggest that pharmacokinetic parameters increase proportionally with dose. SC31 demonstrates a long half-life, as expected for a monoclonal antibody. Outcome of study

• A total of 23 healthy subjects (19 male and 4 female) aged 26 to 52 years were randomised into five cohorts: A1 , A2, A3, A4 and A5.

• Four cohorts received a single dose of SC31 (Dose 1 : 2 mg/kg, Dose 2: 5 mg/kg, Dose 3: 10 mg/kg, and Dose 4: 20 mg/kg), and one cohort received two doses of SC31 at 20 mg/kg: on day 1 and day 2.

• A total of 21 adverse events (AEs) were reported in 14 subjects across all five cohorts. Two AEs occurred prior to SC31 administration.

• One AE of headache, thrombophlebitis on left lower arm and diarrhoea were considered possibly or probably related to SC31 . All AEs were mild in nature.

• No serious adverse events were reported.

• Overall, no serious safety issues were identified.

• 20 mg/kg was confirmed to be the highest safe dose studied in the single ascending dose cohorts. A second dose of 20 mg/kg is safe to be administered.

Example 9: A phase 2/3 study to evaluate the efficacy and safety of SC31 in patients with

COVID-19

The efficacy and safety of SC31 is evaluated in adult patients diagnosed with COVID-19.

The study is an adaptive, prospective, randomised, blinded, placebo-controlled study in adults with active COVID-19 infection. The study will evaluate the safety and efficacy of novel therapeutic agent SC31 in adult patients diagnosed with mild to moderate COVID-19. The study will have 2 arms. Subjects will be randomized to receive either active product SC31 or placebo. The study will evaluate 400 subjects with COVID-19 infection with mild to moderate infections. Subjects will be randomised to the study drug or placebo in a 1 :1 ratio. There will be interim monitoring to allow early stopping for efficacy, or safety.

Each patient recruited into the study will have a screening on Day -1 or Day 1 , dosing on Day 1 , an observation period of at least 2 hours or until clinically stable and able to leave hospital, whichever is greater based on clinical decision of the treating physician. Subjects will be assessed daily if they are hospitalized. Patients will be asked to attend study visits up to Day 29. All subjects will undergo a series of efficacy, safety, and laboratory assessments. Blood samples and oropharyngeal (OP) swabs will be obtained on Day 1 , 3, 5, 8, 11 , 15 and 29 (if able to return to clinic or still hospitalized). Patients will return to the hospital for further out-patient visits only after their community isolation has been ended, and after patients are declared non-infectious by MOH (or relevant agencies according to country protocol). During community isolation out-patient visits will be replaced by telephonic follow- up. Subsequently an end of study visit will be done on Day 29 or by at least one visit after their community isolation is complete. Patients will be followed up telephonically by two weekly visits up to Day 92.

Primary: The overall objective of the study is to evaluate the clinical efficacy of SC31 relative to the control arm in patients with COVID-19.

Secondary: Evaluate the safety of SC31 as compared to the control arm in patients with COVID-

19.

Evaluate additional clinical efficacy parameters of SC31 as compared to the control arm in patients with COVID-19.

Evaluate the immunogenicity of IV administered SC31 in COVID 19 patients.

Exploratory: Evaluate the virologic efficacy of SC31 as compared to the control arm in patients with COVID-19.

Primary: Change from baseline to Day 5 (± 1 day) in SARS-CoV-2 viral load.

Proportion (percentage) of participants who experience progression of the disease as defined by these events by Day 29:

• COVID-19 related mechanical ventilation and/or supplemental oxygen

• COVID-19 related new hospitalization (defined as >24 hours of acute care)

• COVID-19 related emergency room visit, or

• Death

Secondary: Clinical Severity.

Symptom severity:

• Time to symptom resolution

• Proportion of participants demonstrating symptom resolution via the symptom questionnaire on Days 3, 5, 8, 11 , 15 and 29

• Change in symptom score (total of ratings) from baseline to Days 3, 5,

8, 11 , 15 and 29

Virology:

• Percent of subjects with SARS-CoV-2 detectable on Days 3, 5, 8, 11 , 15 and 29.

• Change from baseline to Days 5, 8, 11 , and 15 (± 1 day) in SARS-CoV- 2 viral load.

Ordinal scale: • Clinical improvement rates as determined by ordinal scale on Days 3, 5, 8, 11 , 15 and 29 [Time Frame: Day 1 through Day 29]

• Mean change in the ranking on an ordinal scale from baseline on Days 3, 5, 8, 11 , 15 and 29

Mortality:

• 15-day mortality.

• 28-day mortality.

Safety of the intervention through 28 days of follow-up as compared to the control arm as assessed by:

• Cumulative incidence of serious adverse events (SAEs)

• Cumulative incidence of Grade 3 and 4 adverse events (AEs).

• Discontinuation or temporary suspension of infusions (for any reason).

• Changes in white cell count, haemoglobin, platelets, creatinine, glucose, total bilirubin, ALT, and AST overtime

• Anti-drug antibody titres to SC31

Exploratory: Development of resistance of SARS-CoV-2 in OP sample at days 2, 5, 8, 11 , 15, and 29.

Population pharmacokinetic parameters [Time Frame: Day 1 through Day 92] Treatment groups and duration

Participants will be screened to achieve 400 subjects in the study.

The screening period is 1-7 days and study duration of up to Day 29, for a total of up to 30 days for each subject. Subjects will receive a single dose of the study drug or placebo. Patients will be followed up telephonically up to 91 days. The overall study duration for the combined is up to 32 weeks.

Key inclusion and exclusion criteria

Key inclusion criteria:

• Participants must be >18 years of age, at the time of signing the informed consent

• Have laboratory-confirmed SARS-CoV-2 infection as determined by polymerase chain reaction (PCR), or other commercial or public health assay in any specimen < 72 hours prior to randomisation

• Are negative for SARS-CoV-2 infection as determined by serology assays (seronegative)

• Have mild or moderate COVID-19 illness, as determined by: o Radiographic infiltrates by imaging (chest x-ray, CT scan, etc.), OR o Have one or more mild or moderate COVID-19 symptoms:

■ Fever

■ Cough ■ Sore throat

■ Malaise

■ Headache

■ Muscle pain

■ Gastrointestinal symptoms, or

■ Shortness of breath with exertion o Not requiring mechanical ventilation and/or supplemental oxygen

• Have high risk as defined as patients who meet at least one of the following criteria: o Have a body mass index (BMI) >35 o Have chronic kidney disease o Have diabetes o Have immunosuppressive disease o Are currently receiving immunosuppressive treatment o Are >65 years of age o Are >55 years of age AND have

■ cardiovascular disease, OR

■ hypertension, OR

■ chronic obstructive pulmonary disease/other chronic respiratory disease

• Agrees to the collection of tissue samples (oropharyngeal or nasal swabs) and venous blood per protocol.

• Women of childbearing potential must agree to use at least one primary form of contraception for the duration of the study

Key exclusion criteria:

• Significant allergies to humanised monoclonal antibodies or any drug component

• Have SpO2 < 93% on room air at sea level or PaO2/FiO2 <300, respiratory rate >30 per minute, heart rate >125 per minute

• Require mechanical ventilation or anticipated impending need for mechanical ventilation

• Have hemodynamic instability requiring use of pressors within 24 hours of randomization

• Severe liver disease (e.g. Child Pugh score > C, AST>5 times upper limit)

• Stage 4 severe chronic kidney disease or requiring dialysis (i.e. estimated glomerular filtration rate (eGFR) < 30)

• Have any co-morbidity requiring surgery within <7 days, or that is considered life-threatening within 29 days of randomization

• Have a history of previous SARS-CoV-2 infection as determined by positive SARS-CoV-2 serology test or positive SARS-CoV-2 test prior to the one serving as eligibility for this study

• Receipt of any investigational or approved treatment for COVID-19 (e.g. Remdesivir, Lopinavir, off-label use of other anti-virals, immune-modulatory drugs, compassionate use, or trial related) within the 30 days prior to the time of the screening evaluation • Have received treatment with a SARS-CoV-2 specific monoclonal antibody; convalescent COVID-19 plasma treatment within 3 months or less than 5 half-lives of the investigational product (whichever is longer) prior to the screening visit

• Have participated in a previous SARS-CoV-2 vaccine study or has received the SARS-CoV- 2 vaccine prior to the screening visit

• Have any serious concomitant systemic disease, condition or disorder that, in the opinion of the investigator, should preclude participation in this study

• Pregnancy or breast feeding

Outcome of study

• A total of 400 subjects with confirmed SARS-CoV-2 infection are randomised into cohorts and receive a single dose of the study drug or placebo.

• SARS-CoV-2 viral load is reduced in subjects receiving SC31 by Day 5 (± 1 day).

• Proportion (percentage) of subjects with at least 1 COVID-19-related medical visit is reduced.

• Proportion (percentage) of subjects who experience progression of the disease as defined by the following events is reduced by Day 29: o COVID-19 related mechanical ventilation and/or supplemental oxygen o COVID-19 related new hospitalization (defined as >24 hours of acute care) o COVID-19 related emergency room visit, or o Death.

• Subjects demonstrate reduced 15- or 28-day risk of mortality.

• SC31 is safe for administration to subjects with confirmed SARS-CoV-2 infection.

The study is followed by a larger phase III study involving 1000-1200 patients who receive two doses of SC31 or a placebo. The phase III study includes hospitalised subjects with severe SARS- CoV-2 infection and requiring low flow oxygen. Endpoints include reducing hospitalisation events and reducing the number of subjects who experience progression of the disease.

Claims

Claims:

1. An antigen-binding molecule, optionally isolated, which binds to SARS-CoV-2 spike protein.

2. The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein.

3. The antigen-binding molecule according to claim 1 or claim 2, wherein the antigen-binding molecule inhibits interaction between SARS-CoV-2 spike protein and ACE2.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule inhibits infection of ACE2-expressing cells by SARS-CoV-2.

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule comprises:

(a)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(b)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(c)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(d)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:27 LC-CDR2 having the amino acid sequence of SEQ ID NO:28 LC-CDR3 having the amino acid sequence of SEQ ID NO:29; or

(e)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(f)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(g)

(i) a heavy chain variable (VH) region incorporating the following CDRs:

(ii) a light chain variable (VL) region incorporating the following CDRs:

(h)

(i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:35 HC-CDR2 having the amino acid sequence of SEQ ID NO:54 HC-CDR3 having the amino acid sequence of SEQ ID NO:37; and

(ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:43 LC-CDR2 having the amino acid sequence of SEQ ID NO:44 LC-CDR3 having the amino acid sequence of SEQ ID NO:45.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:55, 17, 1 , 19, 57, 34, 50 or 53; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9, 26 or 42.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises:

(vii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:50; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42; or

(viii) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:53; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:42.

8. The antigen-binding molecule according to any one of claims 1 to 7, wherein the antigen-binding molecule comprises:

125 (vi) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68, and a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:66.

9. The antigen-binding molecule produced by the cell line MCB-115-05, deposited 5 November 2020 as ATCC patent deposit number PTA-126858.

10. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of claims 1 to 9.

11 . An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 10.

12. A cell comprising an antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, or an expression vector or a plurality of expression vectors according to claim 11 .

13. A cell of the cell line designated MCB-115-05, deposited 5 November 2020 as ATCC patent deposit number PTA-126858.

14. A method for producing an antigen-binding molecule which binds to SARS-CoV-2 spike protein, comprising culturing a cell according to claim 12 or claim 13 under conditions suitable for expression of an antigen-binding molecule by the cell.

15. A composition comprising an antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, an expression vector or a plurality of expression vectors according to claim 11 , or a cell according to claim 12 or claim 13, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

16. A composition comprising an antigen-binding molecule according to any one of claims 1 to 9, wherein the composition comprises:

(i) 2 mM to 200 mM histidine, 2% to 20% (w/v) sucrose; 0.001% to 0.1% (w/v) polysorbate- 80, and has a pH 4.0 to 7.0; or

(ii) 2 mM to 200 mM histidine, 1 mM to 100 mM arginine, 2% to 20% (w/v) sucrose; 0.001% to 0.1% (w/v) polysorbate-80, and has a pH 4.0 to 7.0.

(iii) 1 mM to 100 mM acetate, 2% to 20% (w/v) sucrose; 0.001% to 0.1% (w/v) polysorbate-

80, and has a pH 4.0 to 7.0; or

(iv) 2 mM to 200 mM histidine, 1 mM to 50 mM methionine, 2% to 20% (w/v) sucrose;

0.001% to 0.1% (w/v) polysorbate-80, and has a pH 4.0 to 7.0.

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17. The composition according to claim 16, wherein the composition comprises:

18. An antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, an expression vector or a plurality of expression vectors according to claim 11 , a cell according to claim 12, or a composition according to any one of claims 15 to 17, for use in a method of medical treatment or prophylaxis.

19. An antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, an expression vector or a plurality of expression vectors according to claim 11 , a cell according to claim 12, or a composition according to any one of claims 15 to 17, for use in a method of treatment or prevention of a disease caused by infection with SARS- CoV-2.

20. Use of an antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, an expression vector or a plurality of expression vectors according to claim 11 , a cell according to claim 12, or a composition according to any one of claims 15 to 17, in the manufacture of a medicament for use in a method of treatment or prevention of a disease caused by infection with SARS-CoV-2.

21 . A method of treating or preventing a disease caused by infection with SARS-CoV-2, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule according to any one of claims 1 to 9, a nucleic acid or a plurality of nucleic acids according to claim 10, an expression vector or a plurality of expression vectors according to claim

11 , a cell according to claim 12, or a composition according to any one of claims 15 to 17.

22. Use of an antigen-binding molecule according to any one of claims 1 to 9 to inhibit infection of ACE2-expressing cells by SARS-CoV-2.

23. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 9 bound to SARS-CoV-2 spike protein.

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24. A method for detecting SARS-CoV-2 in a sample, comprising contacting a sample containing, or suspected to contain, SARS-CoV-2 with an antigen-binding molecule according to any one of claims 1 to 9, and detecting the formation of a complex of the antigen-binding molecule with SARS-CoV-2 spike protein.

25. A method for diagnosing a disease caused by infection with SARS-CoV-2, comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of claims 1 to 9 and detecting the formation of a complex of the antigen-binding molecule with SARS-CoV-2 spike protein.

26. Use of an antigen-binding molecule according to any one of claims 1 to 9 in a method for detecting, localizing or imaging SARS-CoV-2, or cells infected with SARS-CoV-2.

27. Use of an antigen-binding molecule according to any one of claims 1 to 9 as an in vitro or in vivo diagnostic or prognostic agent.

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