CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to United States Provisional Patent Application No. 63/318,687, filed March 10, 2022, and United States Provisional Patent Application No. 63/290,701 , filed December 17, 2021 , the entire contents of each of which is herein incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (2022-079-03_SL_21 Nov2022.xml; Size: 40,587 bytes; and Date of Creation: November 21 , 2022) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to anti-SARS-CoV-2 specific antibodies and uses thereof. More specifically, the present invention relates to recombinant fusions of antibody fragments that are able to bind and neutralize multiple variants of the SARS-CoV-2 spike protein, and uses thereof in treating human SARS-CoV-2 infections.

BACKGROUND OF THE INVENTION

[0004] The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 caused the coronavirus disease (COVID-19) (Zhou et al. 2020), which led to a worldwide pandemic that three years later includes over 625 million confirmed cases and over 6.5 million associated deaths. By the end of 2021, more than one thousand monoclonal antibodies against SARS-CoV-2 had been reported in the literature (Raybould et al. 2021), with over 200 antibodies in clinical evaluation and 2 antibodies approved for treatment (Yang, Liu, et al. 2020). To maximize neutralization capacity, most of the antibodies in development are directed toward the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein, in order to disrupt interaction between the viral S protein and its host cell receptor ACE2 (Yang, Wang, et al. 2020). These recombinant antibodies block viral entry by binding various epitopes on the RBD in a manner that fundamentally differs from the binding of the S glycoprotein to ACE2 and are therefore susceptible to viral mutational escape in current and emerging variants of SARS-CoV- 2. These variants have already been shown to affect antibody neutralization potencies (Wang, Nair, et al. 2021 ; Wang, Casner, et al. 2021 ; Planas et al. 2021).

[0005] With a view to reducing the risk of mutational escape, there is a need to explore approaches for targeting more conserved epitopes. One way to accomplish this is to identify antibodies that cross-react with different sarbecovi ruses that are phylogenetically more distant than their corresponding variants. In the case of SARS-CoV-2 variants, an opportunity is presented by SARS-CoV-1 , a zoonotic betacoronavirus that caused the 2004 SARS outbreak (Ksiazek et al. 2003), and which also engages ACE2 as a cellular receptor. A maximum likelihood phylogeny analysis of sarbecovirus RBDs indicated that the SARS-CoV-1 clade is distinct from the SARS-CoV-2 clade (Starr et al. 2020). Two anti-RBD SARS-CoV-1 antibodies capable of binding the RBD of SARS-CoV-2, albeit with lower affinity, have been discovered and characterized structurally and functionally. These include the single-domain antibody VHH-72 (Wrapp et al. 2020) and the monoclonal IgG antibody CR3022 (Yuan et al. 2020; Tian et al. 2020); however only VHH-72 was found to be capable of neutralizing the SARS-CoV-2 virus. Structural analysis of VHH-72 bound to SARS-CoV-1 RBD suggested that VHH-72 is able to cross-react with SARS-CoV-1 and SARS-CoV-2 due to its binding to a relatively conserved epitope on the RBD. This epitope does not, however, appear to overlap with the ACE2 binding site on the RBD. Rather, ACE2 would clash with the framework region of VHH-72, as opposed to classical receptor blocking in which the complementarity determining region (CDR) would occupy the ACE2 binding interface. VHH-72 binds to the SARS-CoV-1 RBD through an H-bonding network involving CDR loops 2 and 3, in which backbone groups participate extensively (Wrapp et al. 2020). Although this network is likely to be conserved in the case of SARS-CoV-2 RBD binding, VHH-72 exhibited faster dissociation kinetics and reduced affinity in this case (Wrapp et al. 2020).

SUMMARY OF THE INVENTION

[0006] The present invention relates to anti-SARS-CoV-2 specific antibodies, and specifically to single domain antibodies, and uses thereof. More specifically, the present invention relates to recombinant fusions of antibody fragments that are able to bind and neutralize multiple variants of the SARS-CoV-2 spike protein, and uses thereof in treating human SARS-CoV-2 infections.

[0007] In an embodiment, the present invention provides an isolated, purified or recombinant antibody or antibody fragment that is specific for the spike protein of a SARS-CoV-2 variant, wherein the antibody or antibody fragment comprises a CDR1 sequence of GRTFSEYAMG (SEQ ID NO:1); a CDR2 sequence of TISWSGGXiTYYTDSVKG (SEQ ID NO:2); and a CDR3 sequence of AGX2GX3VVSEWDYDYDY (SEQ ID NO: 3); wherein: Xi is S or M; X ₂ is L or W; and X3 is T or V; including any combinations thereof; with the proviso that when the CDR2 sequence is TISWSGGSTYYTDSVKG (SEQ ID NO:4), then the CDR3 sequence is not AGLGTVVSEWDYDYDY (SEQ ID NO:5), and when the CDR3 sequence is

AGLGTVVSEWDYDYDY (SEQ ID NO: 5), then the CDR2 sequence is not

TISWSGGSTYYTDSVKG (SEQ ID NO:4). [0008] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention binds the spike protein of a SARS-CoV-2 variant with greater affinity and/or slower dissociation constant than a comparable antibody or antibody fragment comprising a CDR1 sequence of SEQ ID NO:1 , a CDR2 sequence of SEQ ID NO:4, and a CDR3 sequence of SEQ ID NO:5.

[0009] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment comprises: a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO:9; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO:5; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:7; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:8; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO:7; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO:8; or a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:9.

[0010] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention is specific for a spike protein of a SARS-CoV-2 variant and comprises a sequence selected from the group consisting of: QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGXiTY YT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGX2GX3VVSEWDYDYDYWGQGT QV TVSS (SEQ ID NO: 10), wherein: Xi is S or M; X2 is L or W; and X3 is T or V; including any combinations thereof; with the proviso that when Xi is S, X2 is not L and X3 is not T ; and when X2 is L and X3 is T, Xi is not S.

[0011] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention comprises an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT

DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTWSEWDYDYDYWGQGT QVT

VSS (SEQ ID NO: 12); QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYY T DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 13);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 14);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 15);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 16);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 17); and

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVVVSEWDYDYDYWGQGTQV T VSS (SEQ ID NO: 18); or a sequence substantially identical thereto. In an embodiment, the antibody or antibody fragment comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0012] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention is a single-domain antibody (sdAb). In an embodiment, the antibody or antibody fragment is in a multivalent display format. In an embodiment, the antibody or antibody fragment is linked to an Fc fragment, or comprised within a polypeptide wherein the antibody or antibody fragment is comprised within an Fc fusion. In an embodiment, the antibody or antibody fragment is linked to a human Fc fragment from human I gG 1 , I gG2 , lgG3 or lgG4.

[0013] In a preferred embodiment, the antibody or antibody fragment of the present invention is linked to a human Fc fragment from human I gG 1 with the D270G mutation. [0014] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention comprises an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTWSEWDYDYDYWGQGTQVT

VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EGPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:21);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTVVSEWDYDYDYWGQGTQV T VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVVVSEWDYDYDYWGQGTQV T VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:23);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTVVSEWDYDYDYWGQGTQV T VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:24);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVVVSEWDYDYDYWGQGTQV T VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:25); QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYY T DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:26); and

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:27); or a sequence substantially identical thereto. In an embodiment, the antibody or antibody fragment comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

[0015] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention neutralizes a cellular infection mediated by a SARS-CoV-2 variant with an IC50 below the concentration of 100 ng/mL.

[0016] In a preferred embodiment, the isolated, purified or recombinant antibody or antibody fragment of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 and neutralizes the cellular infection mediated by the SARS-CoV-2 variant more potently than an antibody or antibody fragment comprising SEQ ID NQ:20. Optionally, the antibody or antibody fragment neutralizes the cellular infection with an IC50 below the concentration of 20 ng/mL.

[0017] The present invention provides a nucleic acid molecule encoding the isolated, purified or recombinant antibody or antibody fragment of the present invention or encoding a polypeptide fusion as described herein, specifically an Fc fusion comprising any of the antibody or antibody fragments provided herein. The present invention also provides a vector comprising a nucleic acid molecule encoding the isolated, purified or recombinant antibody or antibody fragment of the present invention or a polypeptide fusion as described herein, specifically an antibody Fc fusion. [0018] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment is immobilized onto a surface.

[0019] The isolated, purified or recombinant antibody or antibody fragment of the present invention may be linked to a cargo molecule, wherein the cargo molecule is a detectable agent, a therapeutic, a drug, a peptide, a carbohydrate moiety, an enzyme, a cytotoxic agent; one or more liposomes loaded with a detectable agent, a therapeutic, a drug, a peptide, an enzyme, or a cytotoxic agent; or one or more nanoparticles, nanowires, nanotubes, or quantum dots.

[0020] There is provided a composition comprising one or more than one isolated, purified or recombinant antibody or antibody fragment of the present invention and a pharmaceutically- acceptable carrier, diluent, or excipient.

[0021] The present invention provides a method of treating a SARS-CoV-2 infection, comprising administering the isolated, purified or recombinant antibody or antibody fragment to a subject in need thereof.

[0022] In an embodiment there is provided a method of capturing the spike protein of a SARS- CoV-2 variant, comprising contacting a sample with one or more than one isolated, purified or recombinant antibody or antibody fragment and allowing the spike protein of the SARS-CoV-2 variant to bind to the antibody or antibody fragment.

[0023] In an embodiment there is provided a method of detecting a spike protein of a SARS- CoV-2 variant, comprising contacting a sample with one or more than one isolated, purified or recombinant antibody or antibody fragment, allowing the antibody or antibody fragment to bind the spike protein, and detecting the bound antibody or antibody fragment using a suitable detection and/or imaging technology.

[0024] In an embodiment, the isolated, purified or recombinant antibody or antibody fragment is for use to treat a SARS-CoV-2 infection.

[0025] In an embodiment, there is provided a use of the isolated, purified or recombinant antibody or antibody fragment or a composition as described herein to treat a SARS-CoV-2 infection.

[0026] In an embodiment, there is provided a use of the isolated, purified or recombinant antibody or antibody fragment in the manufacture of a medicament for the treatment of a SARS- CoV-2 infection.

[0027] The present invention provides an isolated, purified or recombinant antibody or antibody fragment that is specific to the spike protein of SARS-CoV-2 and comprises a CDR1 sequence of GRTFSEYAMG (SEQ ID NO:1), a CDR2 sequence of TISWSGGX1TYYTDSVKG (SEQ ID NO:2), and a CDR3 sequence of AGX2GX3WSEWDYDYDY (SEQ ID NO: 3); wherein: Xi is S or M; X ₂ is L or W; and X3 is T or V; and combinations thereof; with the proviso that when CDR2 is TISWSGGSTYYTDSVKG (SEQ ID NO:4) then CDR3 is not AGLGTVVSEWDYDYDY (SEQ ID NO: 5), and vice-versa (i.e. when CDR3 is AGLGTVVSEWDYDYDY (SEQ ID NO: 5), CDR2 is not TISWSGGSTYYTDSVKG (SEQ ID NO:4)). The antibody or antibody fragment of the present invention may bind to the spike protein of a SARS-CoV-2 variant with greater affinity and/or a slower dissociation constant than an antibody or antibody fragment comprising a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:5.

[0028] The isolated, purified or recombinant antibody or antibody fragment may comprise: a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 5; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 7; a CDR1 of SEQ ID NO:1 ; a CDR2 of SEQ ID NO:4; and a CDR3 of SEQ ID NO: 8; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 7; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 8; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 9; or a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6; and a CDR3 of SEQ ID NO: 9.

[0029] To summarize, the isolated, purified or recombinant antibody or antibody fragment of the present invention may be selected from the group consisting of: an isolated, purified or recombinant antibody or antibody fragment comprising CDR1 , CDR2 and CDR3 sequences as provided below:

[0030] The present invention also provides an isolated, purified or recombinant antibody or antibody fragment having an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGX 1TYYT

DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGX2GX3VVSEWDYDYDYWG QGTQV TVSS (SEQ ID NO: 10); wherein: Xi is S or M; X2 is L or W; and X3 is T or V; and combinations thereof; but excluding SEQ ID NO: 11.

[0031] An isolated, purified or recombinant antibody or antibody fragment of the present invention having an amino acid sequence of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGX 1TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGX2GX3WSEWDYDYDYWGQGTQ V TVSS (SEQ ID NO: 10); wherein: Xi is S or M; X2 is L or W; and X3 is T or V, with the proviso that when Xi is S, X2 is not L and X3 is not T ; and when X2 is L and X3 is T, Xi is not S.

[0032] Furthermore, the present invention provides an isolated, purified or recombinant antibody or antibody fragment having an amino acid sequence selected from the group consisting of: SEQ ID NOS: 12 to 18; or a sequence substantially identical thereto, for example a sequence having at least 99% sequence identity to any of SEQ ID NOS: 12 to 18.

[0033] The isolated, purified or recombinant antibody or antibody fragment of the present invention may be a single-domain antibody (sdAb); the sdAb may be of camelid origin.

[0034] The isolated, purified or recombinant antibody or antibody fragment of the present invention may be in a multivalent display format. Furthermore, an isolated, purified or recombinant antibody or antibody fragment of the present invention may be linked to an Fc fragment, which may from human lgG1 , lgG2, lgG3 or lgG4. In certain non-limiting examples, an isolated, purified or recombinant antibody or antibody fragment of the present invention may be linked to an engineered human IgG 1 Fc fragment, for example carrying the D270G mutation, for attenuating immune effector functions or other mutations for other purposes including mutations that modulate circulating half-life via FcRn recycling.

[0035] Thus, in preferred non-limiting embodiments, the present invention also provides an isolated, purified or recombinant antibody or antibody fragment having an amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 27; or any sequence substantially identical thereto.

[0036] The isolated, purified or recombinant antibodies or antibody fragments provided by the present invention neutralize the cellular infection mediated by a SARS-CoV-2 variant with an IC50 below the concentration of 100 ng/mL. In certain embodiments, the isolated, purified or recombinant antibody or antibody fragment can have an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and neutralize the cellular infection caused by a SARS-CoV-2 variant more potently than an antibody or antibody fragment comprising the amino acid sequence of SEQ ID NO:20. Preferably, the antibody or antibody fragment neutralizes the cellular infection with an IC50 below the concentration of 20 ng/mL.

[0037] The isolated or purified antibody or antibody fragment of the present invention may be immobilized onto a surface.

[0038] The isolated, purified, or recombinant antibody or antibody fragment of the present invention may be linked to a cargo molecule; the cargo molecule may be a detectable agent, a therapeutic, a drug, a peptide, a protease, an enzyme, a carbohydrate moiety, or a cytotoxic agent; one or more liposomes loaded with a detectable agent, a therapeutic, a drug, a peptide, an enzyme, or a cytotoxic agent; or one or more nanoparticles, nanowires, nanotubes, or quantum dots.

[0039] The present invention further encompasses a nucleic acid molecule encoding the isolated, purified, or recombinant antibody or antibody fragment as provided in the present invention. The present invention also includes a vector comprising the nucleic acid molecule encoding an antibody or antibody fragment of the present invention.

[0040] Also provided is a composition comprising one or more than one isolated, purified, or recombinant antibody or antibody fragment of the present invention and a pharmaceutically- acceptable carrier, diluent, or excipient.

[0041] The present invention provides engineered agents capable of binding, detecting, capturing, and/or neutralizing SARS-CoV-2. Accordingly, fusion proteins described in this invention and targeting the SARS-CoV-2 spike protein’s receptor binding domain (RBD) may block the SARS-CoV-2 spike protein interaction with the ACE2 receptor on the host cell, thereby preventing virus entry into the host cell; a critical initial step in viral infection and subsequent replication inside the host cell.

[0042] The present invention further provides a method of treating a SARS-CoV-2 infection, comprising administering the isolated, purified, or recombinant antibody or antibody fragment of the present invention or the composition described above to a subject in need thereof.

[0043] In another aspect, there is provided a method of capturing a SARS-CoV-2 spike protein, comprising contacting a sample with one or more than one isolated, purified, or recombinant antibody or antibody fragment of the present invention immobilized onto a surface, and allowing the SARS-CoV-2 spike protein to bind the antibody or antibody fragment. The method just described may further comprise identifying the captured SARS-CoV-2 spike protein, for example by mass spectrometric methods and/or eluting the SARS-CoV-2 spike protein. [0044] The present invention additionally provides a method of detecting a SARS-CoV-2 spike protein, comprising contacting a sample with one or more than one isolated, purified, or recombinant antibody or antibody fragment linked to a cargo molecule, allowing the one or more than one isolated, purified, or recombinant antibody or antibody fragment linked to the cargo molecule to bind the SARS-CoV-2 spike protein, and detecting the bound antibody or antibody fragment using a suitable imaging or detection technology. The cargo molecule may be a detectable agent.

[0045] Additional aspects and advantages of the present invention will be apparent in view of the following description. The detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the scope of the invention will become apparent to those skilled in the art in light of the teachings of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] These and other features of the invention will now be described by way of example, with reference to the appended drawings:

[0047] FIGURE 1. Amino-acid sequence of VHH-72 with point mutations designed for optimization of SARS-CoV-2 spike protein binding affinity shown in bold. Kabat numbering is shown on top. FIGURES 2A and 2B. Characterization of Protein A-purified lead mutants of VHH- 72-Fc by analytical UPLC-SEC (FIG. 2A) and SDS-PAGE staining (FIG. 2B). In both figures, lane 2 is S56M.L97W (SEQ ID NO: 24), lane 3 is S56M.T99V (SEQ ID NO: 25), lane 4 is L97W.T99V (SEQ ID NO: 26), and lane 5 is S56M,L97W,T99V (SEQ ID NO: 27).

[0048] FIGURES 3A, 3B, 3C, and 3D. Biophysical testing of VHH-72-Fc mutant hits. FIG. 3A: Dissociation rates (fa) determined by SPR for the three single-mutant hits and resulting multiple mutants, for binding to immobilized spike RBDs from SARS-CoV-1 , SARS-CoV-2 Wuhan, SARS- CoV-2 Beta B.1.351 variant and SARS-CoV-2 Delta B.1.617.2 variant. FIG. 3B: Overlaid SPR sensorgrams for the parental variant and the four multiple mutants for binding to immobilized spike RBD from SARS-CoV-2 Wuhan. FIG. 3C: Overlaid SPR sensorgrams for the parental variant and the triple mutant for binding to immobilized spike RBD from SARS-CoV-2 B.1.1.529 (Omicron). FIG. 3D: Overlaid DSC thermograms for the parental variant and the four multiple mutants.

[0049] FIGURES 4A and 4B. Viral neutralization efficacy by VHH-72-Fc mutant leads. FIG. 4A: Neutralization of VLPs expressing the SARS-CoV-2 Wuhan spike protein for infecting HEK293T cells co-expressing human ACE2 and TMPRSS2. FIG. 4B: Neutralization of VLPs expressing the SARS-CoV-2 Delta variant B.1.617.2 spike protein for infecting HEK293T cells co-expressing human ACE2 and TMPRSS2.

[0050] FIGURE 5. Neutralization of live SARS-CoV-2 Wuhan virus for infecting VERO-E6 cells by VHH-72-Fc mutant leads.

[0051] FIGURES 6A and 6B. In vivo therapeutic efficacy of VHH-72-Fc mutant leads in SARS- CoV-2 infected hamsters. Male hamsters were challenged intranasally with 10 ⁴ PFU of SARS- CoV-2 Wuhan isolate. Four hours later they were administered test articles intraperitoneally at a dose of 10 mg/kg. (FIG. 6A: Body weight changes during the time course of the experiment. FIG. 6B: Live virus titers in lung tissues at day 5 post-infection determined with the plaque assay. Data plotted as mean +/- standard error.

[0052] FIGURES 7A and 7B. Structural details of designed VHH-72 mutations. FIG. 7A: Structural context of the mutations corresponding to the 3 single-mutant hits (black Ca spheres labeled) of the VHH-72 (black cartoon). Current SARS-CoV-2 mutations of concern are indicated and labeled on the spike RBD (gray surface). The relative position of the ACE2 receptor ectodomain (light-gray Ca trace) bound to the SARS-CoV-2 spike RBD is taken form the PDB entry 6M17. FIG 7B: Detailed interactions at the three designed mutation sites. The VHH-72 rendering is in dark-gray cartoon, and the spike RBD rendering is in light-gray cartoon.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention relates to anti-SARS-CoV-2 specific antibodies and uses thereof. More specifically, the present invention relates to recombinant fusions of antibody fragments that bind and neutralize multiple variants of the SARS-CoV-2 spike protein, and uses thereof in treating human SARS-CoV-2 infection.

[0054] The term “COVID-19” as used herein refers to coronavirus disease 2019, a disease caused by the SARS-CoV-2 virus. Symptoms of COVID include respiratory tract infections such as lower respiratory tract infections, high fever, dry cough, shortness of breath, pneumonia; gastro-intestinal symptoms, such as diarrhea; organ failure (kidney failure and renal dysfunction); septic shock and death in severe cases.

[0055] The term “SARS-CoV-2” as used herein refers to severe acute respiratory syndrome virus 2 (SARS-CoV-2), and variants thereof, which was identified as the cause of a serious outbreak starting in Wuhan, China, and which has rapidly spread to other areas of the globe. The term “variant” as used herein refers to a strain of the SARS-CoV-2 virus having one or more mutations, whether naturally occurring or engineered, relative to another variant of the SARS- CoV-2 virus. For example, a variant may have one or more mutations in the spike protein relative to the spike protein of the originally identified SARS-CoV-2 virus (the Wuhan strain). Non limiting examples of SARS-CoV-2 variants include the alpha (B.1.1.7), beta (B.1.351 , B.1.351.1 ,B.1.351.2, B.1.351.3, B.1.351.4), delta (B.1.617.2, AY.1 , AY.2, AY.3, AY.3.1), gamma (P.1 , P.1.1 , P.1.2), and omicron (B.1.1.529) variants.

[0056] As used herein the term “isolated”, when used in reference to an antibody or antibody fragment, means that the antibody or antibody fragment has been removed from the genetic environment in which it was generated or expressed. For example, an “isolated” antibody or antibody fragment may be removed from an organism or host cell in which the antibody or antibody fragment was expressed.

[0057] As used herein the term “purified” when used in reference to an antibody or antibody fragment, relates to enrichment of the antibody or antibody fragment relative to other components present at the time of antibody production or expression. For example, the antibody or antibody fragment may be enriched relative to cellular components, other proteins, and/or other molecules that were present when the antibody or antibody fragment was generated or expressed. Absolute purity is not required for an antibody or antibody fragment to be considered “purified”. Some impurities may still be present. For example, an antibody or antibody fragment may be considered to be “purified” if it has a purity of 60% or greater.

[0058] As used herein, the term “recombinant”, when used in relation to an antibody or antibody fragment, means that the antibody or antibody fragment has been produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed antibody or antibody fragment is inserted into a suitable expression vector that is in turn introduced into a host cell to allow expression of the recombinant antibody or antibody fragment. A recombinant antibody or antibody fragment may include amino acid sequences from two or more sources, such as different proteins or different species. A recombinant antibody or antibody fragment may also include one or more synthetic amino acid sequences.

[0059] As used herein the term “comparable”, when used in reference to an antibody or antibody fragment, means that the antibody or antibody fragment is the same as the reference antibody or antibody fragment, except at the specified amino acid positions. Put another way, the only difference between the “comparable” antibody or antibody fragment and the reference antibody or antibody fragment is the presence of amino acid substitution(s) in the comparable antibody or antibody fragment relative to the reference antibody or antibody fragment, at one or more of the given amino acid positions.

[0060] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. [0061] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0062] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.

[0063] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least 5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0064] More specifically, the term “about” means plus or minus 0.1 % to 50%, 5% to 50%, 10% to 40%, 10% to 20%, or 10% to 15%, preferably 5% to 10%, most preferably about 5% of the number to which reference is being made. The term “about” may also be used to mean within the error margin for a method or instrument used to collect a measurement, within technical tolerance for manufacturing, or allowing a certain degree of variation around a given value, provided the functionality is still present. The term “about” also allows for rounding to the nearest significant figure.

[0065] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

[0066] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0067] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0068] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure, are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0069] Here, using 3D-structural information, the present inventors optimized VHH-72 by applying ADAPT (Assisted Design of Antibody and Protein Therapeutics), a platform that interleaves structure-based computational predictions with experimental testing in order to optimize the binding affinity of a biologic for its target (Vivcharuk et al. 2017). New in this work is the dual-affinity optimization, also known as selectivity optimization, between antibody bonding to multiple targets. Hence, we aimed to optimize the weak binding of VHH-72 to SARS-CoV-2 spike protein while retaining or even improving the strong binding to SARS-CoV-1 spike protein. Past applications of ADAPT affinity maturation have led to 10-100-fold binding affinity improvements of several antibody Fab and VH fragments (Vivcharuk et al. 2017; Sulea et al. 2018). More recently, ADAPT was also applied for optimizing antibody binding selectivity towards the acidic environment of solid tumors relative to the physiological pH around normal cells (Sulea et al. 2020). ADAPT achieves these levels of affinity and selectivity optimization by significant focusing of the vast mutation space available. Depending on the size of the system, only a few dozen protein variants typically need to be produced, purified and tested. Lead designed mutants of VHH-72 were formatted as fusions with a human lgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for in vitro virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for other SARS-CoV-2 variants currently in circulation. Lead variants were tested in the hamster model of SARS-CoV-2 infection and afforded marked therapeutic benefits in terms of recovery from the SARS-CoV-2 infection with corresponding reductions of viral titers, while also demonstrating improvements relative to the efficacy of the parental VHH72-Fc molecule. These improved VHH-72 mutants are predicted to establish novel interactions with the spike protein antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of SARS-CoV-2 infections and associated organ injuries caused by current and potentially emerging SARS-CoV-2 variants.

[0070] As used herein, the terms “variant” and “SARS-CoV-2 variant” refer to a strain of the SARS-CoV-2 virus that has one or more mutations in its spike protein relative to the spike protein of the Wuhan strain of SARS-CoV-2 (GenBank accession no. YP_009724390.1 , as described in Wu et al, 2020). The variant may be, but need not be, a variant of SARS-CoV-2 that has been designated by the World Health Organization as a variant of interest or a variant of concern. [0071] The present invention provides engineered single-domain antibodies (VHHS) capable of binding to the SARS-CoV-2 spike protein and neutralizing the virus. Accordingly, fusion proteins described in this invention are capable of targeting the SARS-CoV-2 spike protein’s receptor binding domain (RBD) and consequently blocking the SARS-CoV-2 spike protein interaction with the human ACE2 receptor on the host cell, thereby preventing virus entry into the host cell; a critical initial step in viral infection and subsequent replication inside the host cell.

[0072] As used herein, the term “specific for”, when used in relation to binding by an antibody or antibody fragment, relates to the ability of the antibody or antibody fragment to discriminate between the desired target (e.g. SARS-CoV-2 spike protein) and an unrelated target, such as a host cell protein. An antibody or antibody fragment specific for the spike protein of a SARS-CoV- 2 variant may specifically bind the SARS-CoV-2 spike protein of multiple SARS-CoV-2 variants, including the spike protein of the first-identified (Wuhan) strain of SARS-CoV-2. It may further specifically bind related spike proteins, such as the spike protein of SARS-CoV-1. An antibody or antibody fragment may be considered to be specific for its target if it binds the target with greater affinity than it binds unrelated proteins, allowing the target protein to be detected above background levels of binding. Binding specificity is a relative property and can be determined, for example, using a technique such as surface plasmon resonance (SPR), western blotting, or enzyme-linked immunosorbent assay (ELISA). In a particular, non-limiting, embodiment, an antibody or antibody fragment as described herein may be considered to be specific for a spike protein of a SARS-CoV-2 variant if it binds the spike protein with a dissociation constant (KD) or a dissociation rate (fa), that is equal to (i.e. , no statistically significant difference from, using a p- value of 0.05) or lower than that of VHH-72 when assayed under the same, or substantially the same, conditions.

[0073] Thus, the present invention provides an isolated, purified or recombinant antibody or fragment comprising a CDR1 sequence of GRTFSEYAMG (SEQ ID NO:1); a CDR2 sequence of TISWSGGXiTYYTDSVKG (SEQ ID NO:2); and a CDR3 sequence of AGX2GX3VVSEWDYDYDY (SEQ ID NO: 3); wherein: Xi is S or M; X2 is L or W; and X3 is T or V; and combinations thereof; and with the proviso that when CDR2 is TISWSGGSTYYTDSVKG (SEQ ID NO:4) then CDR3 is not AGLGTVVSEWDYDYDY (SEQ ID NO: 5), and vice-versa (i.e. CDR2 is not TISWSGGSTYYTDSVKG (SEQ ID NO:4) when CDR3 is AGLGTVVSEWDYDYDY (SEQ ID NO: 5); and wherein the antibody or antibody fragment is specific to the spike protein of SARS-CoV- 2. The antibody or antibody fragment of the present invention binds to SARS-CoV-2 spike protein with greater affinity than an antibody or antibody fragment comprising a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:5.

[0074] The isolated, purified or recombinant antibody or antibody fragment may comprise: a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 9; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 5; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 7; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 8; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 7; a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:6, and a CDR3 of SEQ ID NO: 8; or a CDR1 of SEQ ID NO:1 , a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 9.

[0075] The term “antibody”, also referred to in the art as “immunoglobulin” (Ig), used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while the heavy chain folds into a variable (VH) and three constant (CH, CH2, CHS) domains. Interaction of the heavy and light chain variable domains (VH and L) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.

[0076] The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (VH) and light (VL) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. The Kabat definition of the “complementarity-determining regions” (CDR) is based on sequence variability at the antigenbinding regions of the VH and VL domains (Kabat and Wu 1991). The Chothia definition of the “hypervariable loops” (H or L) is based on the location of the structural loop regions in the VH and VL domains (Chothia and Lesk 1987). As these individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. For this reason, the regions forming the antigen-binding site are presently referred to herein as CDR L1, CDR L2, CDR L3, CDR H1 , CDR H2, CDR H3 in the case of antibodies comprising a VH and a VL domain; or as CDR1 , CDR2, CDR3 in the case of the antigen-binding regions of either a heavy chain or a light chain. The CDR/loops can also be referred to according to the IMGT numbering system (Lefranc et al. 2003), which was developed to facilitate comparison of variable domains. Additionally, a standardized delimitation of the framework regions and of the CDR is provided.

[0077] An “antibody fragment” as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of VL and H connected with a peptide linker), Fab, F(ab’)2, single domain antibody (sdAb; a fragment composed of a single VL or VH), and multivalent presentations of any of these.

[0078] In a non-limiting example, the antibody fragment may be an sdAb derived from a naturally-occurring source. Heavy chain antibodies of camelid origin (Hamers-Casterman et al. 1993) lack light chains and thus their antigen binding sites consist of one domain, termed VHH. sdAbs have also been observed in shark and are termed VNAR (Nuttall et al. 2003). Other sdAbs may be engineered based on human Ig heavy and light chain sequences (Jespers et al. 2004). As used herein, the term “sdAb” includes those sdAbs directly isolated from VH, VHH, VL, or VNAR reservoir of any origin through phage display or other technologies, sdAbs derived from the aforementioned sdAbs, recombinantly produced sdAbs, as well as those sdAbs generated through further modification of such sdAbs by humanization, affinity maturation, stabilization, solubilization, e.g., camelization, or other methods of antibody engineering. Also encompassed by the present invention are sdAb homologues, derivatives, or fragments that retain the antigenbinding function and specificity of the sdAb.

[0079] A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDRs may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDRs of the sdAb or variable domain are referred to herein as CDR1 , CDR2, and CDR3, and delineated according to the Kabat definition for CDR2 and CDR3, and by the union of Kabat and Chothia definitions for CDR1 (Chothia and Lesk 1987; Kabat and Wu 1991). Also, the Kabat numbering system is used throughout (FIG. 1).

[0080] In an embodiment, an antibody or antibody fragment of the invention may be an sdAb. The sdAb may be of camelid origin or derived from a camelid VHH, and thus may be based on camelid framework regions; alternatively, the CDR described above may be grafted onto VNAR, VHH, H or L framework regions. In yet another alternative, the hypervariable loops described above may be grafted onto the framework regions of other types of antibody fragments (Fv, scFv, Fab). The present embodiment further encompasses an antibody fragment that is “humanized” using any suitable method known in the art including, for example but not limited to, CDR grafting and veneering. Humanization of an antibody or antibody fragment comprises replacing one or more amino acids in the sequence with its/their human counterpart(s), as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or antibody fragment when introduced into human subjects. In the process of CDR grafting, one or more than one heavy chain CDR as defined herein may be fused or grafted to a human variable region ( H, or VL), or to other human antibody fragment framework regions (Fv, scFv, Fab). In such a case, the conformation of said one or more than one hypervariable loop is preserved, and the affinity and specificity of the sdAb for its target (i.e., SARS-CoV-2 spike protein) is also preserved. CDR grafting is known in the art and is described in at least the following: US Patent No. 6180370, US Patent No. 5693761 , US Patent No. 6054297, US Patent No. 5859205, and European Patent No. 626390. Veneering, also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of the antibody or antibody fragment; thus, buried non-humanized residues, which may be important for CDR conformation, are preserved while the potential for immunological reaction against solvent- exposed regions is minimized. Veneering is known in the art and is described in at least the following: US Patent No. 5869619, US Patent No. 5766886, US Patent No. 5821123, and European Patent No. 519596. Persons of skill in the art would also be amply familiar with methods of preparing such humanized antibody fragments and humanizing amino acid positions.

[0081] In a specific, non-limiting example, the antibody or antibody fragment provided herein is a pan-specific anti-SARS-CoV-2 antibody capable of binding the spike protein of a plurality of SARS-CoV-2 variants and may comprise an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGX iTYY TDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGX2GX3WSEWDYDYDYWGQGT Q VTVSS (SEQ ID NO: 10); wherein: Xi is S or M; X2 is L or W; and X3 is T or V; and combinations thereof; provided that when Xi is S, X2 is not L and X3 is not T ; or when X2 is L and X3 is T, Xi is not S.

The present invention accordingly provides an isolated, purified or recombinant antibody or antibody fragment having an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 12);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 13);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 14);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 15);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 16);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 17); and

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT VSS (SEQ ID NO: 18); or a sequence substantially identical thereto.

[0082] A sequence substantially identical thereto may have at least 65%, at least 70%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the reference sequence, or have any percent identity or fractional percent sequence identity between 65% and 100% (e.g., 84%, 67.7% or 96.85%) to the reference sequence. [0083] A substantially identical sequence may comprise one or more conservative amino acid mutations relative to the reference sequence. It is known in the art that one or more conservative amino acid mutations relative to a reference sequence may yield a mutant polypeptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. A conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity).

[0084] In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid" it is meant hydrophilic amino acids having a side chain pKa value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include arginine (Arg or R) and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gin or Q) and histidine (His or H). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale (Eisenberg et al. 1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Vai or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pKa value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

[0085] Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at http://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art. [0086] The substantially identical sequences of the present invention may have at least 65% sequence identity to the reference sequence; in another example, the substantially identical sequences may have at least 65%, at least 70%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or any percentage or fractional percentage therebetween, at the amino acid level or at the nucleotide sequence level to one or more sequences described herein. Importantly, the substantially identical sequence retains the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity at the amino acid level may be due to conservative amino acid mutation(s). In another non-limiting example, the present invention may be directed to an antibody or antibody fragment comprising a sequence having at least 98% or at least 99% sequence identity to that of a VHH described herein.

[0087] An isolated, purified or recombinant antibody or antibody fragment of the present invention may bind to a conformational or linear epitope. A conformational epitope is formed by amino acid residues that are discontinuous in sequence, but proximal in the three-dimensional structure of the antigen. In contrast, a linear epitope (also referred to in the art as a “sequential epitope”) is recognized by its linear amino acid sequence, or primary structure. The conformational and linear epitopes of the antibodies or antibody fragments of the present invention recognize conformational and linear epitopes located in the region of TcdA responsible for cell-receptor binding.

[0088] The antibody or antibody fragment of the present invention may also comprise one or more additional sequences to aid in expression, detection or purification of a recombinant antibody or antibody fragment. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or antibody fragment may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc or a Hiss or Hise), or a combination of any two or more thereof. In another example, the one or more additional sequences may comprise a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, one or more linker sequences may be used in conjunction with the one or more additional sequences or tags, or may serve as a detection/purification tags.

[0089] The antibody or antibody fragment of the present invention may also be in a multivalent display format, also referred to herein as multivalent presentation. Multimerization may be achieved by any suitable method known in the art. For example, and without wishing to be limiting in any manner, multimerization may be achieved using self-assembly molecules as described in Zhang, Li, et al. 2004; Zhang, Tanha, et al. 2004; and W02003/046560. The described method produces pentabodies by expressing a fusion protein comprising the antibody or antibody fragment of the present invention and the pentamerization domain of the B-subunit of an AB5 toxin family (Merritt and Hol 1995); the pentamerization domain assembles into a pentamer, through which a multivalent display of the antibody or antibody fragment is formed. Each subunit of the pentamer may be the same or different, and may have the same or different specificity. Additionally, the pentamerization domain may be linked to the antibody or antibody fragment using a linker; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules, but should not hamper the antigen-binding properties of the antibody.

[0090] Other forms of multivalent display are also encompassed by the present invention. For example, and without wishing to be limiting, the antibody or antibody fragment may be presented as a dimer, a trimer, or any other suitable oligomer. This may be achieved by methods known in the art, for example direct linking connection, c-jun/Fos interaction (de Kruif and Logtenberg 1996) or “knobs-into-holes” interaction (Ridgway, Presta, and Carter 1996). Another method known in the art for multimerization is to dimerize the antibody or antibody fragment using an Fc domain, e.g., human Fc domains. The Fc domains may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to lgG1 , lgG2, etc. In this approach, the Fc gene in inserted into a vector along with the sdAb gene to generate a sdAb-Fc fusion protein (Bell et al. 2010; Iqbal et al. 2010); the fusion protein is recombinantly expressed then purified. For example, and without wishing to be limiting in any manner, multivalent display formats may encompass chimeric formats of VHHS linked to an Fc domain, or bi- or tri-specific antibody fusions with two or three VHHS recognizing unique epitopes. Enhanced viral neutralizing efficacy may also be obtained using various techniques, including PEGylation, fusion to serum albumin, or fusion to serum albumin-specific antibody fragments; these approaches increase their blood circulation half-lives, size and avidity.

[0091] Certain non-limiting embodiments of the present invention may incorporate a human lgG1-Fc fragment including a one-residue (alanine) linker, followed by the P226-P243 hinge region and the A244-G477 CH2 and CH3 domains (Kabat numbering was used throughout). The C233 in the hinge region, normally used to link to the light chain of a conventional human lgG1 antibody, can be optionally mutated to serine in order to prevent formation of undesired covalent disulfide-bonded adducts. We introduced a D270G mutation in the CH2 domain in order to attenuate immune receptor functions by reducing binding to human Fey receptors (Shields et al. 2001). Attenuation, rather than complete abrogation of immune receptor functions, would reduce antibody-dependent enhancement (ADE) (Bournazos, Gupta, and Ravetch 2020), as well as reduce the risk exacerbating the hyperinflammatory response often associated with severe COVID-19 development (Manson et al. 2020) while still benefitting from the natural pathogen clearance mechanism of macrophages.

[0092] Therefore, the present invention provides fusion proteins, specifically Fc fusion proteins, comprising an isolated, purified or recombinant antibody or antibody fragment of the present invention, wherein the fusion protein provided may comprise an amino acid sequence selected from the group consisting of:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTWSEWDYDYDYWGQGTQVT

X/SSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEGPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:21);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTWSEWDYDYDYWGQGTQVT VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVWSEWDYDYDYWGQGTQVT

VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EGPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSL TCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:23);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGTWSEWDYDYDYWGQGTQVT VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDG VEVHNAKTKPREEQ YNSTYR VVS VL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQ VYTLPPSRDEL TKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:24);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGVWSEWDYDYDYWGQGTQVT

X/SSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEGPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSL TCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:25);

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT X/SSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEG PEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:26); and

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGM TYYT DSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGWGVWSEWDYDYDYWGQGTQVT VSSAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGP EVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISK AKGQPREPQ VYTLPPSRDEL TKNQ VSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:27); or a sequence substantially identical thereto.

[0093] The isolated, purified or recombinant antibody or antibody fragment of the present invention possesses the ability to neutralize a cellular infection by a SARS-CoV-2 variant. Preferably, this inhibition of infection is achieved at low concentrations which preferably correspond to IC50 values below 100 ng/mL. The isolated, purified or recombinant antibody or antibody fragment may have an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 and neutralizes the cellular infection caused by SARS-CoV-2 more potently than an antibody or antibody fragment comprising SEQ ID NQ:20, and preferably with an IC50 below the concentration of 20 ng/mL. Importantly, in certain embodiments, the isolated, purified or recombinant antibody or antibody fragment of the present invention is advantageously pan-specific, that is to say capable of neutralizing multiple variants of the SARS-CoV-2, for example, and not limited to, Wuhan, Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) virus variants. This broad-spectrum neutralization is achievable due to a similarly broad-spectrum capacity of the antibody or antibody fragment of the present invention to bind to the spike protein of various SARS-CoV-2 variants.

[0094] The present invention also encompasses nucleic acid sequences encoding the molecules as described herein. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms or host cells. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described. [0095] The present invention further encompasses the isolated, purified or recombinant antibody or antibody fragment immobilized onto a surface using various methodologies; for example, and without wishing to be limiting, the antibody or antibody fragment may be linked or coupled to the surface via His-tag coupling, biotin binding, covalent binding, adsorption, and the like. Immobilization of the antibody or antibody fragment of the present invention may be useful in various applications for capturing, purifying or isolating proteins. The solid surface may be any suitable surface, for example, but not limited to the well surface of a microtiter plate, channels of surface plasmon resonance (SPR) sensorchips, membranes, beads (such as magnetic-based or sepharose-based beads or other chromatography resin), glass, plastic, stainless steel, a film, or any other useful surface such as nanoparticles, nanowires and cantilever surfaces.

[0096] Thus, the present invention also provides a method of capturing and detecting the presence of the spike protein of a SARS-CoV-2 variant, comprising contacting a sample (such as, but not limited to SARS-CoV-2 infected human/animal organ or tissue sample fluid, or any other suitable sample) with one or more than one isolated, purified or recombinant antibody or antibody fragment of the present invention. The isolated, purified or recombinant antibody or antibody fragment may be immobilized onto a surface. The SARS-CoV-2 spike protein then binds to the isolated, purified, or recombinant antibody or antibody fragment and is thus captured. The SARS-CoV-2 spike protein may then optionally be identified by mass spectrometric methods and/or released or eluted from its interaction with the antibody or antibody fragment. Methods for releasing or eluting bound molecules are well-known to those of skill in the art (for example but not limited to heat elution steps), as are spectrometric methods capable of detecting and identifying the SARS-CoV-2 spike protein. The isolated, purified, or recombinant antibody or antibody fragment of the present invention allows for the use of particularly robust affinity purification reagents due to its resistance to acidic and heat elution steps.

[0097] The invention also encompasses the antibody or antibody fragment as described above linked to a cargo molecule. The cargo molecule may be a detectable agent, a therapeutic agent, a drug, a peptide, an enzyme, a protease, a carbohydrate moiety, a cytotoxic agent, one or more liposomes loaded with any of the previously recited types of cargo molecules, or one or more nanoparticles, nanowires, nanotubes, or quantum dots or any suitable molecule or any biological or chemical moiety. For example, and without wishing to be limiting in any manner, the cargo molecule may be a protease that may digest the SARS-CoV-2 spike protein; in a further nonlimiting example, the protease may be linked to a VHH such as a mutant VHH that is protease resistant. In yet another non-limiting example, the cargo molecule may be a cytotoxic agent that may be antiviral or toxic towards host cells “infected” with SARS-CoV-2. In a further non-limiting example, the cargo molecule is a liposome, which makes the construct well-suited as a delivery agent for mucosal vaccines. The cargo molecule may be linked to the antibody or antibody fragment by any suitable method known in the art. For example, and without wishing to be limiting, the cargo molecule may be linked to the peptide by a covalent bond or ionic interaction. The linkage may be achieved through a chemical cross-linking reaction, or through fusion using recombinant DNA methodology combined with any peptide expression system, such as bacteria, yeast or mammalian cell-based systems. Methods for linking an antibody or antibody fragment to a therapeutic agent or detectable agent would be well-known to a person of skill in the art.

[0098] The present invention also encompasses an antibody or antibody fragment linked to a detectable agent. For example, the SARS-CoV-2 spike protein-specific antibody or antibody fragment may be linked to a radioisotope, a paramagnetic label, a fluorophore, an affinity label (for example biotin, avidin, etc), fused to a detectable protein-based molecule, nucleotide, quantum dot, nanoparticle, nanowire, or nanotube or any other suitable agent that may be detected by imaging methods. In a specific, non-limiting example, the antibody or antibody fragment may be linked to a fluorescent agent such as FITC or may genetically be fused to the Enhanced Green Fluorescent Protein (EGFP). The antibody or antibody fragment may be linked to the detectable agent using any method known in the art (recombinant technology, chemical conjugation, etc.).

[0099] Thus, the present invention further provides a method of detecting a SARS-CoV-2 spike protein, comprising contacting a sample (such as, but not limited to a SARS-CoV-2 infected human/animal organ or tissue, sample fluid, or any other suitable sample) with one or more than one isolated, purified or recombinant antibody or antibody fragment of the present invention. The isolated, purified or recombinant antibody or antibody fragment may be linked to a detectable agent. The SARS-CoV-2 spike protein can then be detected using detection and/or imaging technologies known in the art, such as, but not limited to mass spectrometric or immunoassay methods.

[0100] For example, and without wishing to be limiting in any manner, the isolated, purified or recombinant antibody or antibody fragment linked to a detectable agent may be used in immunoassays (IA) including, but not limited to enzyme IA (EIA), ELISA, “rapid antigen capture”, “rapid chromatographic IA”, and “rapid EIA”. For examples, see Turgeon, Novicki et al. 2003, Musher, Manhas et al. 2007, Russmann, Panthel et al. 2007, Fenner, Widmer et al. 2008, Planche, Aghaizu et al. 2008, Sloan, Duresko et al. 2008).

[0101] The present invention also encompasses a composition comprising one or more than one isolated, purified or recombinant antibody or antibody fragment as described herein. The composition may comprise a single antibody or antibody fragment as described above, or it may comprise a mixture of antibodies or antibody fragments. [0102] The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, be compatible with the method of delivery of the composition, and not be deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (for example, but limited to lyophilised or encapsulated), capsule or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody or antibody fragment. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be so formulated as to deliver the antibody or antibody fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time-release, or other suitable technologies for delivery of the antibody or antibody fragment. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.

[0103] The present invention also comprises a method of treating a SARS-CoV-2 infection, comprising administering the isolated, purified or recombinant antibody or antibody fragment of the present invention, or a composition comprising the antibody or antibody fragment, to a subject in need thereof. Any suitable method of delivery may be used. For example, and without wishing to be limiting in any manner, the antibody or antibody fragment, or the composition, may be delivered systemically (orally, nasally, intravenously, intraperitoneally, intramuscularly, etc.) or may be delivered to the gastrointestinal tract. Those of skill in the art would be familiar with such methods of delivery.

[0104] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0105] The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner. [0106] Example 1: In silico affinity maturation

[0107] The 2.2-A crystal structure of the VHH-72 sdAb bound to the RBD of the SARS-CoV-1 spike protein was downloaded from the Protein Data Bank (PDB ID: 6WAQ) (Wrapp et al. 2020), and was used as template to building a homology model of the VHH-72 sdAb bound to the RBD of the SARS-CoV-2 spike protein (Wuhan sequence). Both complexes were refined by constrained energy minimization using the AMBER force field (Hornak et al. 2006; Cornell et al. 1995), with a distance-dependent dielectric and an infinite distance cutoff for non-bonded interactions. Non-hydrogen atoms were restrained at their crystallographic positions with harmonic force constants of 40 and 10 kcal/(mol A ² ) for the backbone and side-chain atoms, respectively, except for the amino-acid residues mutated in the SARS-CoV-2 homology model that were allowed to move freely. Resulting structures of the two complexes were used as starting points for tandem affinity maturation of the VHH-72. The ADAPT platform was then used for affinity maturation by single-point scanning mutagenesis simulations carried out at several positions within the CDR loops of VHH-72, as described previously for other systems (Vivcharuk et al. 2017; Sulea et al. 2018). Briefly, we used three protocols, SIE-SCWRL (Krivov, Shapovalov, and Dunbrack 2009; Naim et al. 2007; Sulea and Purisima 2012), FoldX (Guerois, Nielsen, and Serrano 2002; Schymkowitz et al. 2005), and Rosetta (O Conchuir et al. 2015; Rohl et al. 2004) for building the structures and evaluating the energies of single-point mutations to 17 other possible natural amino acids (Cys and Pro were excluded) at these positions of the parental sequence. A consensus approach over specific versions of these three protocols was applied for building and scoring the VHH-72 mutants. Scoring of binding affinity was mainly based on the average Z-score and also on the average rank score over the scores calculated with the three component energy functions, SIE (Naim et al. 2007; Sulea and Purisima 2012), FoldX-FOLDEF (Guerois, Nielsen, and Serrano 2002), and Rosetta- Interface (O Conchuir et al. 2015). Further technical and implementation details of this consensus approach and its component methods can be found in Sulea et al (Sulea et al. 2016). Prior to binding affinity predictions, the FoldX-FOLDEF energy function (Guerois, Nielsen, and Serrano 2002) was used to estimate the effect of substitutions on the internal stability of the VHH structure. Thus, mutations predicted to be destabilizing by introducing folding free energy changes larger than 2.71 kcal/mol (i.e. , 100-fold increase of unfolding equilibrium constant) relative to the parental molecule were discarded from further evaluation. Selection of designed mutants for experimental testing took into account the consensus Z-scores obtained in both complexes undergoing optimization. Preferred criteria for selecting a particular mutation required an improvement in binding to SARS-CoV-2 RBD with a consensus Z-score below -1 together with an improvement in binding to SARS-CoV-1 RBD with a consensus Z-score below 0. [0108] In the first round of ADAPT, 476 single-point mutations to all natural amino acids except Cys and Pro were computationally evaluated in the CDRs of VHH-72. The scanned region covered 28 positions (S30-E31 from CDR1 , S52-K64 from CDR2, and G96-D100g from CDR3) that when substituted have the potential to alter the antigen-binding affinity.

[0109] The selection of the most likely single-point mutants with improved antigen-binding affinities was primarily guided by the best ADAPT consensus Z-scores to both CoV-1 and CoV-2 RBDs. The imposed score-based criteria were stricter towards improving binding affinity for CoV- 2, for which the parental antibody had weaker binding than for CoV-1. Hence, single mutants were first selected to have consensus Z-score < -1.0 for CoV-2 and < 0 for CoV-1. This dual-score based selection retained 25 mutations at 9 positions in the CDR loops 2 and 3 (Table 1). A second selection step was then applied to retain some of the best scoring mutations at each position, in addition to reducing redundancy in the physico-chemical nature of the mutation for cases where multiple mutants were proposed at a given position. Visual inspection of structural model was involved to prune mutations with apparent poor steric and electrostatic complementarity, as in the case of mutations at position T57, and selection of the lower scoring mutation at position T99. The final list proposed for experimental testing in the first round included a 13 single mutations at 8 positions, which were well balanced between CDR loops 2 (6 mutations at 4 positions) and 3 (7 mutations at 4 positions). This is a desired attribute in the set of single mutants that will increase the likelihood of additive contributions of mutation effects in the subsequent round of combining successful mutations. The list included single mutations S53W, G55M, S56H, S56M, D61 R and D61 F from CDR2 and L97W, T99V, V100Y, V100I, V100aM, V100aL and V100al from CDR3.

[0110] Table 1. ADAPT Consensus Z-scores used for VHH-72 mutation selection.

Z-score Selected

UUrx r osinon MUiailOn . , . _ . . _ .

CoV-2 CoV-1 Round 1 Round 2

W -1.7 -1.6 +

S53

Y -1.0 -0.8

M -1.8 -1.6 +

G55 W -1.2 -1.2

L -1.1 -1.1

H -3.2 -3.3 +

„ M -2.4 -2.2 + +

2

S56 Q - ¹ - ³ - ¹ - ³

V -1.1 -1.1

L -1.1 -1.1

F -1.0 -1.3

H -1.2 -1.1

T57 W -1.2 -1.1

D61 R -1.7 -1.4 + -1.6 -1.4 -2.3 -1.6 + + -1-2 -1.0 -1.0 -0.9 + + -1.9 -1.5 + -1.5 -1.6 -1.4 -0.8 + -2.6 -2.3 + -2.2 -2.3 + -1.4 -1.4 + -1.4 -1.2

[0111] Example 2: Protein expression and purification

[0112] These 13 designed single-point mutants of VHH-72 were produced recombinantly alongside the parental VHH-72 as fusions with human lgG1-Fc fragment including a one-residue (alanine) linker, followed by the P226-P243 hinge region and the A244-G477 CH2 and CH3 domains (Kabat numbering was used throughout). The C233 in the hinge region, normally used to link to the light chain of a conventional human lgG1 antibody, was mutated to serine in order to prevent formation of undesired covalent disulfide-bonded adducts. We also introduced the D270G mutation in the CH2 domain in order to attenuate immune receptor functions by reducing binding to human Fey receptors (Shields et al. 2001). It would be expected that this attenuation, rather than complete abrogation of immune receptor functions, would reduce antibody-dependent enhancement (ADE) (Bournazos, Gupta, and Ravetch 2020), as well as reduce the risk exacerbating the hyperinflammatory response often associated with severe COVID- 19 development (Manson et al. 2020), while still benefitting from the natural pathogen clearance mechanism of macrophages.

[0113] The DNA sequences of parental and mutated VHH-72 variants fused to human IgG 1 Fc carrying the D270A mutation, codon-optimized for CHO cells, were synthesized and cloned into the pTT5™ plasmid by GenScript (Piscataway, NJ). Productions were carried out by transient transfection of CHO-55E1 cells as described previously (Stuible et al. 2021), at scales between 25 mL and 500 mL. Briefly, cells were transfected at a viable cell density of ~6x10 ⁶ /mL with 1.4 pug of total DNA and 10 ig PEI MAX (Polysciences, Inc., Warrington, PA) per mL. Transfected DNA consisted of a 8.5:1 .0:0.5 ratio of VHH-72-Fc, Bcl-XL (anti-apoptotic) and GFP (transfection marker) expression vectors, respectively. After addition of DNA: PEI polyplexes, cell cultures were incubated for 24 h on an orbital shaking platform at an agitation rate of 120 rpm at 37°C in a humidified 5% CO2 atmosphere. Cultures were then fed with Feed 4 (Fujifilm Irvine Scientific, Santa Ana CA) at 2.5% v/v and anti-clumping supplement (Fujifilm Irvine Scientific) at 1 :500, and transferred to 32°C. Cultures were supplemented again with Feed 4 (5% v/v) at 5 days-post- transfection and then harvested at day 7. Cell density and viability were determined by direct counting of cell samples with a Cedex™ automated cell counting system (Roche Innovatis, Bielefeld, Germany) using the trypan blue dye exclusion method. Post-transfection cell densities remained between 0.5-2x10 ⁷ cells/mL with viability greater than 89%. Titers ranged between 351- 528 mg/L among the VHH-72-Fc variants.

[0114] Cell cultures were harvested by centrifugation at 20 minutes at 4,000 rpm and supernatants were then filtered using 0.2 urn Stericup™ or Steriflip™ vacuum filtration units (MilliporeSigma, Burlington MA). Purifications from cell-culture supernatants were performed by protein-A affinity chromatography, using 1-, 5- or 10-mL MabSelect™ SuRe™ columns (Cytiva Life Sciences) depending on production scale. Columns were equilibrated in HyClone™ Dulbecco's phosphate-buffered saline (DPBS; Cytiva Life Sciences). Supernatants were loaded at a residence time > 3 min. Columns were washed in DPBS and eluted with 100 mM citrate buffer pH 3.6. Neutralization was done in 10% (v/v) 1 M HEPES. Fractions containing the VHH-72-Fc variants were pooled and the citrate buffer was exchanged against DPBS using Zeba Spin (ThermoFisher Scientific) or HiPrep 26/10 desalting columns (Cytiva Life Sciences), and sterilized by filtration through 0.2 pm filters. Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) was used to assess the purity of all eluates. Homodimer percentage after protein-A affinity purification and desalting was higher than 99% according to UPLC-SEC analysis. Variants used for in vivo studies were concentrated at approximately 1.3 mg/mL or 6.7 mg/mL by ultrafiltration using Vivaspin® Turbo turbo centrifugal concentrator with a membrane molecular weight cut off of 10 kDa (Sartorius) at room temperature following the manufacturer’s instructions. During the process, the protein concentration was monitored on a NanoDrop™ 2000 spectrophotometer (ThermoFisher Scientific) using absorbance at 280 nm and the calculated specific extinction coefficient of each variant. All the formulated samples for in vivo studies have an estimated homodimer percentage higher than 98.5% and endotoxin levels below 0.1 EU/mg. In particular, the lead mutants had excellent developability profiles, including: (1) production yields by transient transfection at 25-mL scale in CHO cells ranging between 350-500 mg/L with cell viabilities over 91 % at day 7 post transfection; and (2) single-step purification by Protein-A affinity chromatography that afforded >99% pure material (by analytical UPLC-SEC and SDS-PAGE/Coomassie™ staining, FIG. 2).

[0115] Example 3: Binding affinity measurements

[0116] Purified VHH-72-Fc variants were analyzed for binding to the spike-RBD domains of SARS-CoV-1, SARS-CoV-2 Wuhan, SARS-CoV-2 B.1.351 , SARS-CoV-2 B.1.617.2 and SARS- CoV-2 B.1.1.529 using a Biacore™ T200 surface plasmon resonance (SPR) instrument (Cytiva, Marlborough, MA). Production and purification of these RBDs were carried out as described elsewhere (Colwill et al. 2021). The S-RBD of SARS-CoV-2 B.1.1.529 residues R319-N542 was produced and purified following the same protocols. Samples were assayed at 25°C using PBS containing 0.05% Tween® 20 (Teknova, Hollister, CA) with added 3.4 mM ethylenediaminetetraacetic acid (EDTA) and 0.05% Tween 20 as running buffer. Spike-RBD samples from SARS-CoV2 (Wuhan and B.1.351 variants) and SARS-CoV-1 were diluted to 10 pg/mL in 10 mM NaOAc immobilization buffer pH 4.5 (Cytiva, Marlborough, MA) and immobilized to approximately 350 Rlls using the Immobilization Wizard for NHS/EDC amine coupling within the BiaControl software. The spike-RBD interactions were assessed using single cycle kinetics analysis for each variant with three concentrations using a 10-fold dilution from the top concentration of 100 nM. The VHH-72-Fc samples were injected at 50 pL/min with a contact time of 60 s and a 600-s dissociation. Sensorgrams were double referenced to the mock-activated blank sensor surface and analyzed for kinetic determination using a 1 :1 binding model in BiaEvaluation software v3.1 (GE Healthcare). All VHH-72-Fc variants were run in triplicate. Due to avidity effects from the bivalent nature of the VHH-72-Fc variants, only dissociation rates ( _d ) are reported.

[0117] Binding of purified VHH-72-Fc proteins to CoV-1 and CoV-2 spike protein receptor binding domains (RBDs) was carried out by surface plasmon resonance (SPR). Three of the designed single mutants, T99V and L97W from CDR3 and S56M from CDR2 (FIG. 1) showed improved binding to the CoV-2 spike RBD domain immobilized on the SPR sensorchip, which was primarily due to reduced dissociation rates, _d (Table 2). The largest effect was seen for T99V, which showed a 10-fold reduced _d relative to the parental VHH-72-Fc, while the smallest improvement was seen for the S56M (FIG. 3A). The same trend of improvements was obtained for the South-African variant (Beta, B.1.351) CoV-2 spike RBD albeit the absolute _d values were larger. These 3 single mutants retained the strong binding of the parental VHH-72-Fc to the CoV- 1 spike RBD, which was even slightly improved in the case of the T99V mutant. This was expected since the FoldX folding stability scores were used to filter out mutations predicted to be significantly detrimental with respect to thermal stability.

[0118] Table 2. Binding and stability data of single-mutation hits and multiple mutants of VHH- 72- Fc. k _d (10 ⁴ s’ ¹ ) from spike-RBD

VHH-72-Fc CoV-2 variant CoV-1 B.1.351 B.1.617.2 B.1.1.529

Wuhan .

_ (Beta) _ (Delta) (Omicron) _ Parent 2.1 ± 0.4 91 ± 10 290 ± 100 51 ± 2 > 10 ⁵ 63.0 83.2

S56M 2.5 ± 0.3 31 ± 9 130 ± 10 10 ± 0.6 > 10 ⁵ 64.0 83.2 L97W 1.8 ± 0.3 18 ± 6 79 ± 7 8.1 ± 0.3 > 10 ⁵ 62.0 83.5

T99V 1.2 ± 0.2 7.2 ± 0.1 77 ± 6 6.2 ± 0.2 > 10 ⁵ 63.5 83.6

7000 ±

S56M,L97W 2.0 ± 0.2 4.4 ± 0.2 25 ± 20 3.9 ± 0.04 „ _ 62.6 83.5

1000

S56M,T99V 1.6 ± 0.1 4.1 ± 0.1 6.8 ± 0.3 3.5 ± 0.05 252 ± 3 63.9 83.4

L97W,T99V 0.8 ± 0.1 3.5 ± 0.1 5.6 ± 0.2 1.6 ± 0.02 610 ± 50 62.4 83.6

S56M,L97W,T99V 1.3 ± 0.1 2.8 ± 0.1 3.9 ± 0.1 2.3 ± 0.04 70 ± 1 63.5 83.6

[0119] The three lead single-point mutations from round 1 of ADAPT were carried forward to round 2 of affinity maturation. All possible combination of these mutations were used to generate three double mutants and one triple mutants. Their binding sensorgrams determined by SPR measurements are shown FIG. 3B. All four multiple mutants show improved binding with reduced dissociation rates from the SARS-CoV-2 spike RBD for both the original Wuhan variant and the Beta variant B.1.351 relative to the parent VHH-72-Fc antibody, and also relative to the component single mutants (FIG. 3A and Table 2). The best variant was the triple mutant S56M,L97W,T99V with ka improved over 30-fold for the Wuhan CoV-2 spike RBD, over 70-fold for Beta B.1.351 CoV-2 spike RBD and over 20-fold for Delta B.1.617.2 CoV-2 spike RBD relative to the parent. These dissociation rates, which are in the single-digit 10' ⁴ s ^-1 range, approached that measured against the CoV-1 spike RBD which was maintained between the triple mutant and the parent. However, the double mutant L97W.T99V closely followed the triple mutant with a similar binding strength against the two CoV-2 virus variants, while also affording a 4-fold improved _d from the CoV-1 relative to the parent. This suggests that the double mutant already harbors most of the improvements seen with the triple mutant, in agreement with the single mutation data showing that the S56M mutation affords the smallest improvement among the three progressed mutations. Moreover, parental VHH-72-Fc and single mutants had very low dissociation constants to SARS-CoV-2 B.1.1.529 (Omicron) S RBD (Table 2). Remarkably, the multiple mutants were able to rescue binding, with the best level attained by the triple mutant S56M,L97W,T99V that reached a k _d similar to that of the parental antibody against the Wuhan S- RBD variant (Fig 3C and Table 2). Apparently, the negative impact of mutations in the B.1.1.529 (Omicron) S RBD has been in part offset by the combined new interactions established by the three mutations in the antibody triple mutant. This further demonstrates the generality of the dualaffinity optimization approach against SARS coronaviruses from different clades for broad targeting of virus variants within a given clade.

[0120] Example 4: Thermal stability measurements

[0121] Differential scanning calorimetry (DSC) was used to determine the thermal transition midpoints (T _m ) of the parental and mutant VHH-72-Fc variants. DSC was carried out in a VP- Capillary DSC system instrument (Malvern Instruments Ltd, Malvern, UK). Samples were diluted in DPBS buffer to a final concentration of 0.4 mg/mL. DPBS blank and sample scans were carried out by increasing the temperature from 20°C to 100°C at a rate of 60°C/h, with feedback mode/gain set at “low”, filtering period of 8 s, pre-scan time of 3 min, and under 70 psi of nitrogen pressure. All data were analyzed with Origin 7.0 software (OriginLab Corporation, Northampton, MA). Thermograms were corrected by subtraction of corresponding DPBS blank scans and normalized to the protein molar concentration. The T _m values were determined using a manual fit to two transitions.

[0122] Determination of the melting temperatures by DSC for the three single mutants with improved binding against SARS-CoV2 indicated similar thermal stabilities relative to parental VHH-72-Fc (Table 2). The multiple mutants also advantageously had high thermal stability characterized by VH-domain melting temperature (T _m 1) around 63°C, similar to the parental construct (FIG. 3D and Table 2).

[0123] Example 5: Virus neutralization assays

[0124] Pseudotyped SARS-CoV2 spike lentiviral particles were produced using either the pHDM-SARS-CoV-2 Wuhan-Hu-1 expressing the SARS-CoV-2 Wuhan-Hu-1 spike protein (GenBank # NC_045512) or pcDNA3.3-SARS2-B.1 .617.2 expressing the SARS-CoV-2 B.1 .617.2 (Delta variant) spike protein, a gift from David Nemazee (Addgene plasmid # 172320) under a CMV promoter and packaged into lentiviral vectors obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit, NR-52948 and according to the protocols and reagents described by the Bloom lab (Crawford et al. 2020), with the following modifications: (1) HEK293SF-3F6 cells (Cote et al. 1998) were used for large-scale production of lentiviral particles in 300 mL; (2) post-transfection HEK293SF-3F6 cells were incubated at 33°C for improved yield; (3) 72 h post-infection lentiviral particles were harvested and subjected to concentration by sucrose cushion centrifugation. Briefly, the supernatant was placed on 20% sucrose cushion and spun for 3 h at 37,000xg at 4°C. The pellet containing the concentrated pseudo typed VLP was then resuspended in DMEM with 10% FBS and aliquoted. Titration was performed using HEK293T cells overexpressing human ACE2 and TMPRSS2, obtained from BEI Resources repository of ATCC and the NIH (NR-55293). Pseudovirus neutralization assay was performed according to the previously described protocol (Crawford et al. 2020) and was adapted for 384-well plate. Briefly, 3-fold serial dilutions of the VHH-72-Fc samples were incubated with diluted virus at a 1 :1 ratio for 1 h at 37°C before addition to HEK293- ACE2/TMPRSS2 cells. Infectivity was then measured by luminescence readout per well. Bright- Glo luciferase reagent (Promega, E2620) was added to wells for 2 min before reading with a PerkinElmer Envision instrument. 50% inhibitory concentration (IC50) were calculated with nonlinear regression (log[inhibitor] versus normalized response - variable slope) using GraphPad Prism® 8 (GraphPad Software Inc.).

[0125] These binding affinity improvements prompted us to test the best three multiple mutants for their ability to block the entry of SARS-CoV-2 pseudo typed virus like particle (VLP) into cells in vitro. The first set of experiments employed a non-replicating pseudovirus neutralization assay (Crawford et al. 2020). In this assay, two different VLPs were produced and tested, containing SARS-CoV-2 spike protein from either the Wuhan or Delta B.1.617.2 strains packaged, a luciferase reporter and the minimal set of lentiviral proteins required to assemble the VLPs. A monolayer of HEK293T cells co-expressing human ACE2 and human TMPRSS2 were exposed to the VLPs. The ability to block entry of these particles into cells was detected by loss of signal of the luciferase reporter. The advantageous and unexpected benefits of the antibodies provided in the present invention, as shown in FIG. 4AB and listed in Table 3, the three multiple mutants of VHH-72-Fc blocked cellular infection against the two VLPs more potently than the parental compound. The best neutralization potencies were obtained with the triple mutant S56M,L97W,T99V, which displayed IC50 values of 9 ng/mL and 4 ng/mL against Wuhan and Delta B.1.617.2 spike protein pseudotyped VLPs, respectively, which represent 11-fold and 18- fold improvements, respectively, relative to the parent compound. The double mutant L97W.T99V also showed strong neutralization potencies, amounting to 6-fold and 4-fold improvements, respectively, relative to the parental compound.

[0126] Table 3. In vitro SARS-CoV-2 neutralization efficacy data of VHH-72-Fc lead mutants.

IC50 (ng/mL)

VHH72-Fc variant Pseudovirus Live Virus

Wuhan B.1.617.2 Wuhan

Parent 20 98 72 43

S56M.T99V 25 34 25 3

L97W.T99V 26 16 17 2

S56M,L97W,T99V 27 9 4 2

[0127] Encouraged by these improvements obtained with the pseudotyped VLP assay, we next assessed the ability of these three multiple mutants to neutralize infection of human VERO-E6 cells by the live replicating authentic virus. This was done with a microneutralization assay. SARS- CoV-2 isolate Canada/GN/VIDO-01/2020 was obtained from the National Microbiology Laboratory (Winnipeg, MB, Canada) and propagated on Vero E6 cells and quantified on Vero cells. Whole viral genome sequencing was carried out to confirm exact genetic identity to original isolate. Passage 3 virus stocks were used. Neutralization activity was determined with the microneutralization assay. In brief, 1:5 serial dilutions of 15 ig of each antibody was carried out in DMEM, high glucose media supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U/rnL penicillin-streptomycin, and 1% heat-inactivated fetal bovine serum. SARS-CoV-2 was added at 125 plaque forming units (PFU) in 1 :1 ratio to each antibody dilution and incubated at 37°C for 1 h. After incubation, Vero E6 cells seeded in 96-well plates were infected with virus/antibody mix and incubated at 37°C in humidified/5% CO2 incubator for 72 h post-infection (hpi). Cells were then fixed in 10% formaldehyde overnight and virus infection was detected with mouse anti-SARS-CoV-2 nucleocapsid antibody (R&D Systems, clone #1035111) and counterstained with rabbit anti-mouse IgG-HRP (Rockland Inc.). Colorimetric development was obtained with o-phenylenediamine dihydrochloride peroxidate substrate (Sigma-Aldrich) and detected on Biotek Synergy H1 plate reader at 490 nm. IC50 was determined from non-linear regression using GraphPad Prism 9 software.

[0128] As shown in FIG. 5 and listed in Table 3, the multiple mutants tested inhibited replication of live virus with IC50 values in the 2-3 ng/mL range, hence affording over 20-fold improved efficacies for the best mutants L97W.T99V and S56M,L97W,T99V relative to the parental VHH- 72-Fc. Taken together, these data demonstrate that ADAPT-guided optimization of the VHH-72 paratope for improved CoV-2 spike protein binding affinity can translate into a marked functional improvements at cellular level.

[0129] Example 6: In vivo hamster infection model

[0130] Male Syrian golden hamsters (81-90 g) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). Animals were maintained at the animal facility of the National Research Council Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were in accordance with regulations and guidelines reviewed and approved in animal use protocol 2020.06 by the NRC Human Health Therapeutics Animal Care Committee. Male hamsters were challenged intranasally with 1 x 10 ⁴ PFU of SARS-CoV-2 isolate Canada/ON/VI DG-01/2020. Four hours postchallenge, animals were administered intraperitoneally 150 pl of antibody at the dose of 10 mg/kg. Daily body weights were determined. Animals were euthanized at 5 days post-challenge and viral load was determined for lung tissues by plaque assay. Plaque assay was performed as previously described (Akache et al. 2021).

[0131] The two mutants that performed best in the in vitro neutralization assays, L97W.T99V and S56M,L97W,T99V were submitted to in vivo efficacy testing as therapeutic agents for neutralizing the SARS-CoV-2 infection. Although prophylactic administration usually leads to more pronounced effects, we opted to test these variants in the more relevant scenario appropriate for their eventual intended use as therapeutic agents. Moreover, intraperitoneal administration incurs a further time delay required for the drug distribution from the ventral area into systemic circulation to finally reaching the lungs of treated animals. With this in vivo study plan, both VHH-72-Fc lead mutants tested, S56M,L97W,T99V and L97W.T99V, had a marked impact on the treated groups relative to the control group (vehicle alone), as indicated by reduced body weight losses (FIG. 6A). In the case of S56M,L97W,T99V, the recovery actually led to body weight gains after day 3 post-challenge, which was superior to the recovery afforded by the parental VHH-72-Fc variant. Furthermore, these changes in body weight were correlated with the live virus titres obtained from the lung samples of the corresponding treatment groups. As shown in FIG. 6B, therapeutic dosing led to one order of magnitude reduction of average virus titre in the lungs animals treated with the parental VHH-72-Fc variant, and two orders of magnitude reduction of average virus titre in the lungs animals treated with the optimized VHH-72-Fc variant S56M,L97W,T99V. The trend seen for the in vivo therapeutic efficacy is supported by the trends observed in the in vitro virus neutralization data presented in Example 5. Overall, these data underscore the therapeutic potential of the structure-based optimized VHH-72-Fc for treating SARS-CoV-2 infection with greater efficacies relative to the parental construct.

[0132] Example 7 Structural interpretation of enhanced affinity and stability

[0133] The three mutating positions of VHH-72 interact with the 17-amino-acid contiguous sequence region 368-LYNSASFSTFKCYGVSP-384 (SEQ ID NO: 32) of the SARS-CoV-2 spike protein RBD. These positions are far away from mutations present in currently-circulating SARS- CoV-2 variants of concern (FIG. 7A), suggesting cross-reactivity with these variants. As virus mutations tend to occur at the interface with the ACE2 host receptor, which is distinct from the VHH-72 binding interface (FIG. 7A), it is likely that mutating any of these three positions of VHH- 72 alone or in combination would also retain binding to emerging virus variants in the future.

[0134] One of the advantages of rational structure-guided affinity maturation is that it helps to understand the structural basis for improvement of binding affinity. The positions of these three single-point mutations of VHH-72 at the binding interface with the spike RBD are shown in FIG. 7B. The mutation L97W (from the CDR3 loop) introduced new van-der-Waals contacts and weak polar contacts with the backbone of loop C379-S383 and nonpolar contacts with the aliphatic ring of P384. The adjacent mutation T99V (also from the CDR3 loop) introduces a new hydrophobic contact with the hydrophobic part of the K378 side-chain. Finally, the S56M mutation (from the CDR2 loop) introduces new hydrophobic contacts with the side-chains of F374 and F377. However, on the negative side, this hydrophobic mutation is also on close proximity of polar backbone atoms of other residues in the region L367-F374, and eliminates an H-bond anchor to the backbone carbonyl of N370. Since all three mutations interact with a contiguous linear epitope and two of the mutations are adjacent within the CDR3 loop, we expected a less-than-optimal additivity of single mutation effects upon combining them into multiple-point mutants, as it was in fact observed experimental (see next section, vide infra).

[0135] In conclusion, structure-based ADAPT-guided affinity maturation helped improve the virus neutralization efficacy of VHH-72-Fc against SARS-CoV-2. This covered the original Wuhan strain as well as other major virus variants of public concern currently in circulation. Simultaneous dual-affinity optimization against sarbetacoronavirus from distinct phylogenetic clades appears as a good strategy towards achieving cross-specificity against virus variants within a given clade. This dual-optimization was feasible mainly due to relatively conserved S-RBD epitope targeted by VHH-72, which is distinct from that targeted by ACE2 that is prone to mutational escape from most neutralizing antibodies currently under development. The results reported here can have implications towards improved biotherapeutics against SARS-CoV-2 infections caused by current and future emerging variants, possibly reaching beyond the SARS-CoV-2 clade.

[0136] Table 4. Sequence listing of CDR loops of VHH-72 variants. [0137] Table 5. Sequence listing of VHH-72 variants.

[0138] Table 6. Sequence listing of VHH-72-Fc variants.

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